Genetic polymorphisms associated with coronary heart disease, methods of detection and uses thereof

ABSTRACT

The present invention is based on the discovery of genetic polymorphisms that are associated with coronary heart disease and in particular stenosis and MI and response to drug treatment. In particular, the present invention relates to nucleic acid molecules containing the polymorphisms, variant proteins encoded by such nucleic acid molecules, reagents for detecting the polymorphic nucleic acid molecules and proteins, and methods of using the nucleic acid and proteins as well as methods of using reagents for their detection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 60/660,322, filed on Mar. 11, 2005, and U.S. provisional applicationSer. No. 60/711,447, filed on Aug. 24, 2005, the contents of which arehereby incorporated by reference in its entirety into this application.

FIELD OF THE INVENTION

The present invention is in the field of coronary heart disease (CHD)and in particular stenosis and myocardial infarction (MI) diagnosis andtherapy. In particular, the present invention relates to specific singlenucleotide polymorphisms (SNPs) in the human genome, and theirassociation with CHD and related pathologies. Based on differences inallele frequencies in the patient population relative to normalindividuals, the naturally-occurring SNPs disclosed herein can be usedas targets for the design of diagnostic reagents and the development oftherapeutic agents, as well as for disease association and linkageanalysis. In particular, the SNPs of the present invention are usefulfor identifying an individual who is at an increased or decreased riskof developing CHD and in particular stenosis and MI, and for earlydetection of the disease, for providing clinically important informationfor the prevention and/or treatment of CHD such as stenosis and MI, forscreening and selecting therapeutic agents and for predicting apatient's response to therapeutic agents. The SNPs disclosed herein arealso useful for human identification applications. Methods, assays,kits, and reagents for detecting the presence of these polymorphisms andtheir encoded products are provided.

BACKGROUND OF THE INVENTION

CHD is defined in the Framingham study as encompassing angina, MI,coronary insufficiency (which is manifested as ischemia, that is,impaired oxygen flow to the heart muscle), stenosis and coronary heartdisease death (Wilson et al., Circulation 97:1837-1847 (1998)). It issometimes recorded through clinical records that indicate the followinginterventions: coronary artery bypass graft, angioplasty and stentplacement in addition to clinical records of MI, angina, or coronarydeath. This latter definition is used in many population based studiesand clinical trials, but it probably misses silent MI events and someunreported angina.

MI (or heart attack) is the most common cause of mortality in developedcountries. The incidence of MI is still high despite currently availablepreventive measures and therapeutic intervention. More than 1,500,000people in the US suffer acute MI each year (many without seeking helpdue to unrecognized MI), and one third of these people die. The lifetimerisk of coronary artery disease events at age 40 years is 42.4% for men(one in two) and 24.9% for women (one in four) (Lloyd-Jones D M; Lancet,1999 353: 89-92).

MI is a multifactorial disease that involves atherogenesis, thrombusformation and propagation. Thrombosis can result in complete or partialocclusion of coronary arteries. The luminal narrowing or blockage ofcoronary arteries reduces oxygen and nutrient supply to the cardiacmuscle (cardiac ischemia), leading to myocardial necrosis and/orstunning. MI, unstable angina, and sudden ischemic death are clinicalmanifestations of cardiac muscle damage. All three endpoints are part ofthe Acute Coronary Syndrome since the underlying mechanisms of acutecomplications of atherosclerosis are considered to be the same.

Atherogenesis, the first step of pathogenesis of MI, is a complexinteraction between blood elements, mechanical forces, disturbed bloodflow, and vessel wall abnormality that results in plaque accumulation.An unstable (vulnerable) plaque was recognized as an underlying cause ofarterial thrombotic events and MI. A vulnerable plaque is a plaque,often not stenotic, that has a high likelihood of becoming disrupted oreroded, thus forming a thrombogenic focus. MI due to a vulnerable plaqueis a complex phenomenon that includes: plaque vulnerability, bloodvulnerability (hypercoagulation, hypothrombolysis), and heartvulnerability (sensitivity of the heart to ischemia or propensity forarrhythmia). Recurrent myocardial infarction (RMI) can generally beviewed as a severe form of MI progression caused by multiple vulnerableplaques that are able to undergo pre-rupture or a pre-erosive state,coupled with extreme blood coagulability.

The current diagnosis of MI is based on the levels of troponin I or Tthat indicate the cardiac muscle progressive necrosis, impairedelectrocardiogram (ECG), and detection of abnormal ventricular wallmotion or angiographic data (the presence of acute thrombi). However,due to the asymptomatic nature of 25% of acute MIs (absence of atypicalchest pain, low ECG sensitivity), a significant portion of MIs are notdiagnosed and therefore not treated appropriately (e.g., prevention ofrecurrent MIs).

MI risk assessment and prognosis is currently done using classic riskfactors or the recently introduced Framingham Risk Index. Both of theseassessments put a significant weight on LDL levels to justify preventivetreatment. However, it is well established that half of all MIs occur inindividuals without overt hyperlipidemia.

Other emerging risk factors of MI are inflammatory biomarkers such asC-reactive protein (CRP), ICAM-1, SAA, TNF α, homocysteine, impairedfasting glucose, new lipid markers (ox LDL, Lp-a, MAD-LDL, etc.) andpro-thrombotic factors (fibrinogen, PAI-1). These markers havesignificant limitations such as low specificity and low positivepredictive value, and the need for multiple reference intervals to beused for different groups of people (e.g., males-females, smokers-nonsmokers, hormone replacement therapy users, different age groups). Theselimitations diminish the utility of such markers as independentprognostic markers for MI screening.

Genetics plays an important role in MI risk. Families with a positivefamily history of MI account for 14% of the general population, 72% ofpremature MIs, and 48% of all MIs (Williams R R, Am J Cardiology, 2001;87:129). In addition, replicated linkage studies have revealed evidenceof multiple regions of the genome that are associated with MI andrelevant to MI genetic traits, including regions on chromosomes 14, 2, 3and 7 (Broeckel U, Nature Genetics, 2002; 30: 210; Harrap S,Arterioscler Thromb Vasc Biol, 2002; 22: 874-878, Shearman A, HumanMolecular Genetics, 2000, 9; 9, 1315-1320), implying that genetic riskfactors influence the onset, manifestation, and progression of MI.Recent association studies have identified allelic variants that areassociated with acute complications of CHD, including allelic variantsof the ApoE, ApoA5, Lpa, APOCIII, and Klotho genes.

Genetic markers such as single nucleotide polymorphisms (SNPs) arepreferable to other types of biomarkers. Genetic markers that areprognostic for MI can be genotyped early in life and could predictindividual response to various risk factors. The combination of serumprotein levels and genetic predisposition revealed by genetic analysisof susceptibility genes can provide an integrated assessment of theinteraction between genotypes and environmental factors, resulting insynergistically increased prognostic value of diagnostic tests.

Thus, there is an urgent need for novel genetic markers that arepredictive of predisposition to CHD such as MI, particularly forindividuals who are unrecognized as having a predisposition to MI. Suchgenetic markers may enable prognosis of MI in much larger populationscompared with the populations that can currently be evaluated by usingexisting risk factors and biomarkers. The availability of a genetic testmay allow, for example, appropriate preventive treatments for acutecoronary events to be provided for susceptible individuals (suchpreventive treatments may include, for example, statin treatments andstatin dose escalation, as well as changes to modifiable risk factors),lowering of the thresholds for ECG and angiography testing, and allowadequate monitoring of informative biomarkers. Moreover, the discoveryof genetic markers associated with MI will provide novel targets fortherapeutic intervention or preventive treatments of MI, and enable thedevelopment of new therapeutic agents for treating MI and othercardiovascular disorders.

Coronary stenosis is the narrowing of coronary arteries by obstructiveatherosclerotic plaques. The coronary arteries supply oxygenated bloodflow to the myocardium. Although mild and moderate coronary stenosis donot impede resting coronary flow, stenosis>30-45% starts to restrictmaximal coronary flow. Severe coronary stenosis (>70% reduction inluminal diameter) causes stable angina (ischemic chest pain uponexertion). Significant stenosis contributes, along with plaque ruptureand thrombus formation, coronary spasm, or inflammation/infection, tounstable angina as well as myocardial infarction. Together witharrhythmia, coronary stenosis is a major factor of sudden cardiacdeaths, as evidenced by its presence in two or more major coronaryarteries in 90% of adult sudden cardiac death victims.

Coronary stenosis is a prevalent disease. Each year in the UnitedStates, 440,000 new cases of stable angina and 150,000 new cases ofunstable angina occur. This year, an estimated 1.1 million Americanswill have a new or recurrent heart attack. These incidences result inover six million individuals in the U.S. living with stable or unstableangina pectoris, a debilitating condition, and over seven millionindividuals in the U.S. living with a history of myocardial infarction.Coronary stenosis is frequently a deadly disease. It is a majorunderlying cause of CHD, which is the single largest cause of death inthe U.S. Over half a million coronary deaths, including 250,000 suddencardiac deaths, occur each year in U.S.

There is, therefore, an unmet need in early diagnosis and prognosis ofasymptomatic coronary stenosis. This need is particularly significantgiven that early diagnosis or prognosis results can significantlyinfluence the course of disease by influencing treatment choices (forexample, those with genetic risks can be treated to modify risk factorssuch as hypertension, diabetes, inactivity, dyslipidemia, etc.),thresholds (e.g., lipid levels used to trigger the use of lipid-loweringdrugs), and goals (e.g., target blood pressure or lipid levels), andpossibly enhance compliance.

Diagnosis of coronary stenosis currently starts by assessing if the riskprofiles (e.g., hypertension, dyslipidemia, family history, diabetes,etc.) and symptoms (e.g., angina) of patients are consistent withcoronary heart disease, followed most commonly by resting and exerciseEKGs. However, risk assessments and EKGs are imperfect diagnostic testsfor stenosis since they can be both insensitive (giving false negatives)and non-specific (giving false positives). Coronary arteriography is thedefinitive test for assessing the severity of coronary stenosis,however, it is not very sensitive in early detection of mild stenosis.It is also an invasive procedure with a small risk of death due to thecatheterization procedure and the contrast dye. Because of this risk, itis typically only used at a time when coronary stenosis is consideredlikely from symptoms or other tests, which is hardly an ideal time tostart intervention.

Coronary stenosis risk is presumed to have a strong genetic component.It is well known that several major risk factors of coronary disease areheritable, e.g. serum lipid levels (Perusse L. et. al., ArteriosclerThromb Vasc Biol (1997): 17(11) 3263-9) and obesity (Rice T. et. al.,Int J Obes Relat Metab Disord (1997):21(11) 1024-31). Indeed, severalknown genetic defects are individually sufficient to cause elevatedserum LDL-cholesterol (e.g., familial hypercholesterolemia) leading topremature coronary disease (Goldstein and Brown, Science 292 (2001):1310-12). In addition, linkage studies in humans have replicated thefindings of the link of several chromosomal regions (quantitative traitloci) to coronary heart disease and related diseases and risk factors(Pajukanta P. et. al., Am J Hum Genet 67 (2000):1481-93, Francke S. et.al., Human Molecular Genetics (2001): 10 (24) 2751-65). Finally, afamily history of premature coronary disease is a significant factor inthe risk assessment and diagnosis of coronary disease (Braunwald E.,Zipes D. and Libby P., Heart Disease, 6^(th) ed. W. B. Saunders Company,2001, 28).

Although many risk factors for coronary stenosis have been identified,including age, diabetes, hypertension, high serum cholesterol, smoking,etc., and genetic factors play significant roles in several of theserisk factors, significant genetic risk factors are likely to exist whichhave not been identified to date. In addition to the anecdotal coronarydisease patients that exhibit few traditional risk factors, a study ofmultiple existing risk factors showed that only half of the“population-attributable risk” was attributable to known risk factors(Change M. et. al., J Clin Epidemiol (2001) 54 (6) 634-44). Therefore,the presently known risk factors are inadequate for predicting coronarystenosis risk in individuals. Given the magnitude of the disease, thereis an urgent need for genetic markers that are predictive of coronarystenosis risk. Such genetic markers could increase the prognosticability of existing risk assessment methods and complement currentdiagnostic methods such as exercise EKG, especially in early detectionof disease when intervention is most effective and should ideally start.

Reduction of coronary and cerebrovascular events and total mortality bytreatment with HMG-CoA reductase inhibitors (statins) has beendemonstrated in a number of randomized, double blinded, placebocontrolled prospective trials (Waters, D. D., Clin Cardiol, 2001. 24(8Suppl): p. III13-7, Singh, B. K. and J. L. Mehta, Curr. Opin. Cardiol.,2002. 17(5): p. 503-11). These drugs have their primary effect throughthe inhibition of hepatic cholesterol synthesis, thereby upregulatingLDL receptor in the liver. The resultant increase in LDL catabolismresults in decreased circulating LDL, a major risk factor forcardiovascular disease.

Statins can be divided into two types according to their physicochemicaland pharmacokinetic properties. Statins such as lovastatin, simvastatin,atorvastatin, and cerevastatin are hydrophobic in nature and, as such,diffuse across membranes and thus are highly cell permeable. Hydrophilicstatins such as pravastatin are more polar, such that they requirespecific cell surface transporters for cellular uptake (Ziegler, K. andW. Stunkel, Biochim Biophys Acta, 1992. 1139(3): p. 203-9, Yamazaki, M.,et al., Am J Physiol, 1993. 264(1 Pt 1): p. G36-44, Komai, T., et al.,Biochem Pharmacol, 1992. 43(4): p. 667-70). The latter statin utilizes atransporter, OATP2, whose tissue distribution is confined to the liverand, therefore, they are relatively hepato-specific inhibitors (Hsiang,B., et al., J Biol Chem, 1999. 274(52): p. 37161-8). The former statins,not requiring specific transport mechanisms, are available to all cellsand they can directly impact a much broader spectrum of cells andtissues. These differences in properties may influence the spectrum ofactivities that each statin possesses. Pravastatin, for instance, has alow myopathic potential in animal models and myocyte cultures comparedto other hydrophobic statins (Masters, B. A., et al., Toxicol ApplPharmacol, 1995. 131 (1): p. 163-74. Nakahara, K., et al., Toxicol ApplPharmacol, 1998. 152(1): p. 99-106, Reijneveld, J. C., et al., PediatrRes, 1996. 39(6): p. 1028-35).

Evidence from gene association studies is accumulating to indicate thatresponses to drugs are, indeed, at least partly under genetic control.As such, pharmacogenetics—the study of variability in drug responsesattributed to hereditary factors in different populations—maysignificantly assist in providing answers toward meeting this challenge(Roses, A. D., Nature, 2000. 405(6788): p. 857-65, Mooser, V., et al., JThromb Haemost, 2003. 1(7): p. 1398-1402, Humma, L. M. and S. G. Terra,Am. J. Health Syst Pharm, 2002. 59(13): p. 1241-52). Numerousassociations have been reported between selected genotypes, as definedby SNPs and other sequence variations and specific responses tocardiovascular drugs. Polymorphisms in several genes have been suggestedto influence responses to statins including CETP (Kuivenhoven, J. A., etal., N Engl J Med, 1998. 338(2): p. 86-93), beta-fibrinogen (de Maat, M.P., et al., Arterioscler Thromb Vasc Biol, 1998. 18(2): p. 265-71),hepatic lipase (Zambon, A., et al., Circulation, 2001. 103(6): p. 792-8,lipoprotein lipase (Jukema, J. W., et al., Circulation, 1996. 94(8): p.1913-8), glycoprotein IIIa (Bray, P. F., et al., Am J Cardiol, 2001.88(4): p. 347-52), stromelysin-1 (de Maat, M. P., et al., Am J Cardiol,1999. 83(6): p. 852-6), and apolipoprotein E (Gerdes, L. U., et al.,Circulation, 2000. 101(12): p. 1366-71, Pedro-Botet, J., et al.,Atherosclerosis, 2001. 158(1): p. 183-93). Some of these variants wereshown to effect clinical events while others were associated withchanges in surrogate endpoints. Thus there is the need for markers andindividuals responsiveness to statins.

SNPs

The genomes of all organisms undergo spontaneous mutation in the courseof their continuing evolution, generating variant forms of progenitorgenetic sequences (Gusella, Ann. Rev. Biochem. 55, 831-854 (1986)). Avariant form may confer an evolutionary advantage or disadvantagerelative to a progenitor form or may be neutral. In some instances, avariant form confers an evolutionary advantage to the species and iseventually incorporated into the DNA of many or most members of thespecies and effectively becomes the progenitor form. Additionally, theeffects of a variant form may be both beneficial and detrimental,depending on the circumstances. For example, a heterozygous sickle cellmutation confers resistance to malaria, but a homozygous sickle cellmutation is usually lethal. In many cases, both progenitor and variantforms survive and co-exist in a species population. The coexistence ofmultiple forms of a genetic sequence gives rise to geneticpolymorphisms, including SNPs.

Approximately 90% of all genetic polymorphisms in the human genome areSNPs. SNPs are single base positions in DNA at which different alleles,or alternative nucleotides, exist in a population. The SNP position(interchangeably referred to herein as SNP, SNP site, SNP locus, SNPmarker, or marker) is usually preceded by and followed by highlyconserved sequences of the allele (e.g., sequences that vary in lessthan 1/100 or 1/1000 members of the populations). An individual may behomozygous or heterozygous for an allele at each SNP position. A SNPcan, in some instances, be referred to as a “cSNP” to denote that thenucleotide sequence containing the SNP is an amino acid coding sequence.

A SNP may arise from a substitution of one nucleotide for another at thepolymorphic site. Substitutions can be transitions or transversions. Atransition is the replacement of one purine nucleotide by another purinenucleotide, or one pyrimidine by another pyrimidine. A transversion isthe replacement of a purine by a pyrimidine, or vice versa. A SNP mayalso be a single base insertion or deletion variant referred to as an“indel” (Weber et al., “Human diallelic insertion/deletionpolymorphisms,” Am J Hum Genet 2002 October; 71(4):854-62).

A synonymous codon change, or silent mutation/SNP (terms such as “SNP,”“polymorphism,” “mutation,” “mutant,” “variation,” and “variant” areused herein interchangeably), is one that does not result in a change ofamino acid due to the degeneracy of the genetic code. A substitutionthat changes a codon coding for one amino acid to a codon coding for adifferent amino acid (i.e., a non-synonymous codon change) is referredto as a missense mutation. A nonsense mutation results in a type ofnon-synonymous codon change in which a stop codon is formed, therebyleading to premature termination of a polypeptide chain and a truncatedprotein. A read-through mutation is another type of non-synonymous.codon change that causes the destruction of a stop codon, therebyresulting in an extended polypeptide product. While SNPs can be bi-,tri-, or tetra-allelic, the vast majority of the SNPs are bi-allelic,and are thus often referred to as “bi-allelic markers,” or “di-allelicmarkers.”

As used herein, references to SNPs and SNP genotypes include individualSNPs and/or haplotypes, which are groups of SNPs that are generallyinherited together. Haplotypes can have stronger correlations withdiseases or other phenotypic effects compared with individual SNPs, andtherefore may provide increased diagnostic accuracy in some cases(Stephens et al. Science 293, 489-493, 20 Jul. 2001).

Causative SNPs are those SNPs that produce alterations in geneexpression or in the expression, structure, and/or function of a geneproduct, and therefore are most predictive of a possible clinicalphenotype. One such class includes SNPs falling within regions of genesencoding a polypeptide product, i.e. cSNPs. These SNPs may result in analteration of the amino acid sequence of the polypeptide product (i.e.,non-synonymous codon changes) and give rise to the expression of adefective or other variant protein. Furthermore, in the case of nonsensemutations, a SNP may lead to premature termination of a polypeptideproduct. Such variant products can result in a pathological condition,e.g., genetic disease. Examples of genes in which a SNP within a codingsequence causes a genetic disease include sickle cell anemia and cysticfibrosis.

Causative SNPs do not necessarily have to occur in coding regions;causative SNPs can occur in, for example, any genetic region that canultimately affect the expression, structure, and/or activity of theprotein encoded by a nucleic acid. Such genetic regions include, forexample, those involved in transcription, such as SNPs in transcriptionfactor binding domains, SNPs in promoter regions, in areas involved intranscript processing, such as SNPs at intron-exon boundaries that maycause defective splicing, or SNPs in mRNA processing signal sequencessuch as polyadenylation signal regions. Some SNPs that are not causativeSNPs nevertheless are in close association with, and therefore segregatewith, a disease-causing sequence. In this situation, the presence of aSNP correlates with the presence of, or predisposition to, or anincreased risk in developing the disease. These SNPs, although notcausative, are nonetheless also useful for diagnostics, diseasepredisposition screening, and other uses.

An association study of a SNP and a specific disorder involvesdetermining the presence or frequency of the SNP allele in biologicalsamples from individuals with the disorder of interest, such as stenosisor MI, and comparing the information to that of controls (i.e.,individuals who do not have the disorder; controls may be also referredto as “healthy” or “normal” individuals) who are preferably of similarage and race. The appropriate selection of patients and controls isimportant to the success of SNP association studies. Therefore, a poolof individuals with well-characterized phenotypes is extremelydesirable.

A SNP may be screened in diseased tissue samples or any biologicalsample obtained from a diseased individual, and compared to controlsamples, and selected for its increased (or decreased) occurrence in aspecific pathological condition, such as pathologies related to CHD andin particular, stenosis and MI. Once a statistically significantassociation is established between one or more SNP(s) and a pathologicalcondition (or other phenotype) of interest, then the region around theSNP can optionally be thoroughly screened to identify the causativegenetic locus/sequence(s) (e.g., causative SNP/mutation, gene,regulatory region, etc.) that influences the pathological condition orphenotype. Association studies may be conducted within the generalpopulation and are not limited to studies performed on relatedindividuals in affected families (linkage studies).

Clinical trials have shown that patient response to treatment withpharmaceuticals is often heterogeneous. There is a continuing need toimprove pharmaceutical agent design and therapy. In that regard, SNPscan be used to identify patients most suited to therapy with particularpharmaceutical agents (this is often termed “pharmacogenomics”).Similarly, SNPs can be used to exclude patients from certain treatmentdue to the patient's increased likelihood of developing toxic sideeffects or their likelihood of not responding to the treatment.Pharmacogenomics can also be used in pharmaceutical research to assistthe drug development and selection process. (Linder et al. (1997),Clinical Chemistry, 43, 254; Marshall (1997), Nature Biotechnology, 15,1249; International Patent Application WO 97/40462, Spectra Biomedical;and Schafer et al. (1998), Nature Biotechnology, 16: 3).

SUMMARY OF TH INVENTION

The present invention relates to the identification of novel SNPs,unique combinations of such SNPs, and haplotypes of SNPs that areassociated with CHD, and in particular stenosis and MI. Thepolymorphisms disclosed herein are directly useful as targets for thedesign of diagnostic reagents and the development of therapeutic agentsfor use in the diagnosis and treatment of CHD, and in particularstenosis and MI, as well as predicting a patient's response totherpeutic agents such as statins.

Based on the identification of SNPs associated with CHD, the presentinvention also provides methods of detecting these variants as well asthe design and preparation of detection reagents needed to accomplishthis task. The invention specifically provides, for example, novel SNPsin genetic sequences involved in stenosis and MI, isolated nucleic acidmolecules (including, for example, DNA and RNA molecules) containingthese SNPs, variant proteins encoded by nucleic acid moleculescontaining such SNPs, antibodies to the encoded variant proteins,computer-based and data storage systems containing the novel SNPinformation, methods of detecting these SNPs in a test sample, methodsof identifying individuals who have an altered (i.e., increased ordecreased) risk of developing CHD based on the presence or absence ofone or more particular nucleotides (alleles) at one or more SNP sitesdisclosed herein or the detection of one or more encoded variantproducts (e.g., variant mRNA transcripts or variant proteins), methodsof identifying individuals who are more or less likely to respond to atreatment (or more or less likely to experience undesirable side effectsfrom a treatment, etc.), methods of screening for compounds useful inthe treatment of a disorder associated with a variant gene/protein,compounds identified by these methods, methods of treating disordersmediated by a variant gene/protein, methods of using the novel SNPs ofthe present invention for human identification, etc.

In Tables 1-2, the present invention provides gene information,references to the identification of transcript sequences (SEQ ID NOS:1-67), encoded amino acid sequences (SEQ ID NOS: 68-134), genomicsequences (SEQ ID NOS: 229-299), transcript-based context sequences (SEQID NOS: 135-228) and genomic-based context sequences (SEQ ID NOS:300-504) that contain the SNPs of the present invention, and extensiveSNP information that includes observed alleles, allele frequencies,populations/ethnic groups in which alleles have been observed,information about the type of SNP and corresponding functional effect,and, for cSNPs, information about the encoded polypeptide product. Theactual transcript sequences (SEQ ID NOS: 1-67), amino acid sequences(SEQ ID NOS: 68-134), genomic sequences (SEQ ID NOS: 229-299),transcript-based SNP context sequences (SEQ ID NOS: 135-228), andgenomic-based SNP context sequences (SEQ ID NOS: 300-504), together withprimer sequences (SEQ ID NOS: 505-783) are provided in the SequenceListing.

In one embodiment of the invention, applicants teach a method foridentifying an individual who has an altered risk for developing CHD,comprising detecting a single nucleotide polymorphism (SNP) in any oneof the nucleotide sequences of SEQ ID NOS: 1-67 and 135-504 in saidindividual's nucleic acids, wherein the SNP is as specified in Table 1and Table 2, respectively, and the presence of the SNP is correlatedwith an altered risk for MI in said individual. In a specific embodimentof the present invention, SNPs that occur naturally in the human genomeare provided as isolated nucleic acid molecules. These SNPs areassociated with CHD, and in particular stenosis and MI such that theycan have a variety of uses in the diagnosis and/or treatment of CHD andrelated pathologies. In an alternative embodiment, a nucleic acid of theinvention is an amplified polynucleotide, which is produced byamplification of a SNP-containing nucleic acid template. In anotherembodiment, the invention provides for a variant protein that is encodedby a nucleic acid molecule containing a SNP disclosed herein.

In yet another embodiment of the invention, a reagent for detecting aSNP in the context of its naturally-occurring flanking nucleotidesequences (which can be, e.g., either DNA or mRNA) is provided. Inparticular, such a reagent may be in the form of, for example, ahybridization probe or an amplification primer that is useful in thespecific detection of a SNP of interest. In an alternative embodiment, aprotein detection reagent is used to detect a variant protein that isencoded by a nucleic acid molecule containing a SNP disclosed herein. Apreferred embodiment of a protein detection reagent is an antibody or anantigen-reactive antibody fragment.

Various embodiments of the invention also provide kits comprising SNPdetection reagents, and methods for detecting the SNPs disclosed hereinby employing detection reagents. In a specific embodiment, the presentinvention provides for a method of identifying an individual having anincreased or decreased risk of developing MI by detecting the presenceor absence of one or more SNP alleles disclosed herein. In anotherembodiment, a method for diagnosis of stenosis or MI by detecting thepresence or absence of one or more SNP alleles disclosed herein isprovided.

The nucleic acid molecules of the invention can be inserted in anexpression vector, such as to produce a variant protein in a host cell.Thus, the present invention also provides for a vector comprising aSNP-containing nucleic acid molecule, genetically-engineered host cellscontaining the vector, and methods for expressing a recombinant variantprotein using such host cells. In another specific embodiment, the hostcells, SNP-containing nucleic acid molecules, and/or variant proteinscan be used as targets in a method for screening and identifyingtherapeutic agents or pharmaceutical compounds useful in the treatmentof CHD, and in particular stenosis and MI.

An aspect of this invention is a method for treating CHD, and inparticular stenosis and MI, in a human subject wherein said humansubject harbors a SNP, gene, transcript, and/or encoded proteinidentified in Tables 1-2, which method comprises administering to saidhuman subject a therapeutically or prophylactically effective amount ofone or more agents counteracting the effects of the disease, such as byinhibiting (or stimulating) the activity of the gene, transcript, and/orencoded protein identified in Tables 1-2.

Another aspect of this invention is a method for identifying an agentuseful in therapeutically or prophylactically treating CHD, and inparticular stenosis and MI, in a human subject wherein said humansubject harbors a SNP, gene, transcript, and/or encoded proteinidentified in Tables 1-2, which method comprises contacting the gene,transcript, or encoded protein with a candidate agent under conditionssuitable to allow formation of a binding complex between the gene,transcript, or encoded protein and the candidate agent and detecting theformation of the binding complex, wherein the presence of the complexidentifies said agent.

Another aspect of this invention is a method for treating CHD such asstenosis and/or MI in a human subject, which method comprises:

(i) determining that said human subject harbors a SNP, gene, transcript,and/or encoded protein identified in Tables 1-2, and

(ii) administering to said subject a therapeutically or prophylacticallyeffective amount of one or more agents counteracting the effects of thedisease such as stating.

Many other uses and advantages of the present invention will be apparentto those skilled in the art upon review of the detailed description ofthe preferred embodiments herein. Solely for clarity of discussion, theinvention is described in the sections below by way of non-limitingexamples.

Description of the Files Contained on the CD-R Named CDR Duplicate Copy1 and CDR Duplicate Copy 2

Each of the the CD-Rs contains the following three files:

1) File SEQLIST_(—)0000010RD.txt provides the Sequence Listing. TheSequence Listing provides the transcript sequences (SEQ ID NOS: 1-67)and protein sequences (SEQ ID NOS: 68-134) as referred to in Table 1,and genomic sequences (SEQ ID NOS: 229-299) as referred to in Table 2,for each CHD-associated gene or genomic region (for intergenic SNPs)that contains one or more SNPs of the present invention. Also providedin the Sequence Listing are context sequences flanking each SNP,including both transcript-based context sequences as referred to inTable 1 (SEQ ID NOS: 135-228) and genomic-based context sequences asreferred to in Table 2 (SEQ ID NOS: 300-504). In addition, the SequenceListing provides the primer sequences from Table 3 (SEQ ID NOS:505-783), which are oligonucleotides that have been synthesized and usedin the laboratory to assay the SNPs disclosed in Tables 4-8 during thecourse of association studies to verify the association of these SNPswith coronary heart disease (CHD). The context sequences generallyprovide 100 bp upstream (5′) and 100 bp downstream (3′) of each SNP,with the SNP in the middle of the context sequence, for a total of 200bp of context sequence surrounding each SNP. File SEQLIST_(—)1572ORD.txtis 1,562 KB in size, and was created on Mar. 13, 2006. A computerreadable format of the sequence listing is also submitted herein on aseparate CDR labeled CRF. The information recorded in the CRF CDR isidentical to the sequence listing as provided on the CDR Duplicate Copy1 and Copy 2.

2) File TABLE 1_(—)000001ORD.txt provides Table 1. File TABLE1_(—)000001ORD.txt is 106 KB in size, and was created on Mar. 13, 2006.

3) File TABLE2_(—)000001ORD.txt provides Table 2. FileTABLE2_(—)000001ORD.txt is 155 KB in size, and was created on Mar. 13,2006.

The material contained on the CD-R labeled CD000001ORD is herebyincorporated by 20 reference pursuant to 37 CFR 1.77(b) (4).

Description of Table 1 and Table 2

Table 1 and Table 2 (both provided on the CD-R) disclose the SNP andassociated gene/transcript/protein information of the present invention.For each gene, Table 1 provides a header containing gene, transcript andprotein information, followed by a transcript and protein sequenceidentifier (SEQ ID), and then SNP information regarding each SNP foundin that gene/transcript including the transcript context sequence. Foreach gene in Table 2, a header is provided that contains gene andgenomic information, followed by a genomic sequence identifier (SEQ ID)and then SNP information regarding each SNP found in that gene,including the genomic context sequence.

Note that SNP markers may be included in both Table 1 and Table 2; Table1 presents the SNPs relative to their transcript sequences and encodedprotein sequences, whereas Table 2 presents the SNPs relative to theirgenomic sequences. In some instances Table 2 may also include, after thelast gene sequence, genomic sequences of one or more intergenic regions,as well as SNP context sequences and other SNP information for any SNPsthat lie within these intergenic regions. Additionally, in either Table1 or 2 a “Related Interrogated SNP” may be listed following a SNP whichis determined to be in LD with that interrogated SNP according to thegiven Power value. SNPs can readily be cross-referenced between allTables based on their Celera hCV (or, in some instances, hDV)identification numbers, and to the Sequence Listing based on theircorresponding SEQ ID NOS.

The gene/transcript/protein information includes:

-   -   a gene number (1 through n, where n=the total number of genes in        the Table)    -   a Celera hCG and UID internal identification numbers for the        gene    -   a Celera hCT and UID internal identification numbers for the        transcript (Table 1 only)    -   a public Genbank accession number (e.g., RefSeq NM number) for        the transcript (Table 1 only)    -   a Celera hCP and UID internal identification numbers for the        protein encoded by the hCT transcript (Table 1 only)    -   a public Genbank accession number (e.g., RefSeq NP number) for        the protein (Table 1 only)    -   an art-known gene symbol    -   an art-known gene/protein name    -   Celera genomic axis position (indicating start nucleotide        position-stop nucleotide position)    -   the chromosome number of the chromosome on which the gene is        located    -   an OMIM (Online Mendelian Inheritance in Man; Johns Hopkins        University/NCBI) public reference number for obtaining further        information regarding the medical significance of each gene    -   alternative gene/protein name(s) and/or symbol(s) in the OMIM        entry

Note that, due to the presence of alternative splice forms, multipletranscript/protein entries may be provided for a single gene entry inTable 1; i.e., for a single Gene Number, multiple entries may beprovided in series that differ in their transcript/protein informationand sequences.

Following the gene/transcript/protein information is a transcriptcontext sequence and (Table 1), or a genomic context sequence (Table 2),for each SNP within that gene.

After the last gene sequence, Table 2 may include additional genomicsequences of intergenic regions (in such instances, these sequences areidentified as “Intergenic region:” followed by a numericalidentification number), as well as SNP context sequences and other SNPinformation for any SNPs that lie within each intergenic region (suchSNPs are identified as “INTERGENIC” for SNP type).

Note that the transcript, protein, and transcript-based SNP contextsequences are all provided in the Sequence Listing. The transcript-basedSNP context sequences are provided in both Table 1 and also in theSequence Listing. The genomic and genomic-based SNP context sequencesare provided in the Sequence Listing. The genomic-based SNP contextsequences are provided in both Table 2 and in the Sequence Listing. SEQID NOS are indicated in Table 1 for the transcript-based contextsequences (SEQ ID NOS: 135-228); SEQ ID NOS are indicated in Table 2 forthe genomic-based context sequences (SEQ ID NOS: 300-504).

The SNP information includes:

-   -   context sequence (taken from the transcript sequence in Table 1,        the genomic sequence in Table 2) with the SNP represented by its        IUB code, including 100 bp upstream (5′) of the SNP position        plus 100 bp downstream (3′) of the SNP position (the        transcript-based SNP context sequences in Table 1 are provided        in the Sequence Listing as SEQ ID NOS: 135-228; the        genomic-based SNP context sequences in Table 2 are provided in        the Sequence Listing as SEQ ID NOS: 300-504).    -   Celera hCV internal identification number for the SNP (in some        instances, an “hDV” number is given instead of an “hCV” number).    -   The corresponding public identification number for the SNP, the        RS number.    -   SNP position (position of the SNP within the given transcript        sequence (Table 1) or within the given genomic sequence (Table        2)).    -   “Related Interrogated SNP” is as the interrogated SNP with which        the listed SNP is in LD at the given value of Power.    -   SNP source (may include any combination of one or more of the        following five codes, depending on which internal sequencing        projects and/or public databases the SNP has been observed in:        “Applera”=SNP observed during the re-sequencing of genes and        regulatory regions of 39 individuals, “Celera”=SNP observed        during shotgun sequencing and assembly of the Celera human        genome sequence, “Celera Diagnostics”=SNP observed during        re-sequencing of nucleic acid samples from individuals who have        a disease, “dbSNP”=SNP observed in the dbSNP public database,        “HGBASE”=SNP observed in the HGBASE public database, “HGMD”=SNP        observed in the Human Gene Mutation Database (HGMD) public        database, “HapMap”=SNP observed in the International HapMap        Project public database, “CSNP”=SNP observed in an internal        Applied Biosystems (Foster City, Calif.) database of coding SNPS        (cSNPs)). Note that multiple “Applera” source entries for a        single SNP indicate that the same SNP was covered by multiple        overlapping amplification products and the re-sequencing results        (e.g., observed allele counts) from each of these amplification        products is being provided.    -   Population/allele/allele count information in the format of        [population1(first_allele,count|second_allele,count)population2(first_allele,count|second_allele,count)        total (first_allele,total count|second_allele,total count)]. The        information in this field includes populations/ethnic groups in        which particular SNP alleles have been observed        (“cau”=Caucasian, “his”=Hispanic, “chn”=Chinese, and        “afr”=African-American, “jpn”=Japanese, “ind”=Indian,        “mex”=Mexican, “ain”=“American Indian, “cra”=Celera donor,        “no_pop”=no population information available), identified SNP        alleles, and observed allele counts (within each population        group and total allele counts), where available [“−” in the        allele field represents a deletion allele of an        insertion/deletion (“indel”) polymorphism (in which case the        corresponding insertion allele, which may be comprised of one or        more nucleotides, is indicated in the allele field on the        opposite side of the “|”); “−” in the count field indicates that        allele count information is not available]. For certain SNPs        from the public dbSNP database, population/ethnic information is        indicated as follows (this population information is publicly        available in dbSNP): “HISP1”=human individual DNA (anonymized        samples) from 23 individuals of self-described HISPANIC        heritage; “PAC1”=human individual DNA (anonymized samples) from        24 individuals of self-described PACIFIC RIM heritage;        “CAUC1”=human individual DNA (anonymized samples) from 31        individuals of self-described CAUCASIAN heritage; “AFR1”=human        individual DNA (anonymized samples) from 24 individuals of        self-described AFRICAN/AFRICAN AMERICAN heritage; “P1”=human        individual DNA (anonymized samples) from 102 individuals of        self-described heritage; “PA130299515”; “SC_(—)12_A”=SANGER 12        DNAs of Asian origin from Corielle cell repositories, 6 of which        are male and 6 female; “SC_(—)12_C”=SANGER 12 DNAs of Caucasian        origin from Corielle cell repositories from the CEPH/UTAH        library. Six male and 6 female; “SC_(—)12_AA”=SANGER 12 DNAs of        African-American origin from Corielle cell repositories 6 of        which are male and 6 female; “SC_(—)95_C”=SANGER 95 DNAs of        Caucasian origin from Corielle cell repositories from the        CEPH/UTAH library; and “SC_(—)12_CA”=Caucasians—12 DNAs from        Corielle cell repositories that are from the CEPH/UTAH library.        Six male and 6 female.

Note that for SNPs of “Applera” SNP source, genes/regulatory regions of39 individuals (20 Caucasians and 19 African Americans) werere-sequenced and, since each SNP position is represented by twochromosomes in each individual (with the exception of SNPs on X and Ychromosomes in males, for which each SNP position is represented by asingle chromosome), up to 78 chromosomes were genotyped for each SNPposition. Thus, the sum of the African-American (“afr”) allele counts isup to 38, the sum of the Caucasian allele counts (“cau”) is up to 40,and the total sum of all allele counts is up to 78.

Note that semicolons separate population/allele/count informationcorresponding to each indicated SNP source; i.e., if four SNP sourcesare indicated, such as “Celera,” “dbSNP,” “HGBASE,” and “HGMD,” thenpopulation/allele/count information is provided in four groups which areseparated by semicolons and listed in the same order as the listing ofSNP sources, with each population/allele/count information groupcorresponding to the respective SNP source based on order; thus, in thisexample, the first population/allele/count information group wouldcorrespond to the first listed SNP source (Celera) and the thirdpopulation/allele/count information group separated by semicolons wouldcorrespond to the third listed SNP source (HGBASE); ifpopulation/allele/count information is not available for any particularSNP source, then a pair of semicolons is still inserted as aplace-holder in order to maintain correspondence between the list of SNPsources and the corresponding listing of population/allele/countinformation.

-   -   SNP type (e.g., location within gene/transcript and/or predicted        functional effect) [“MIS-SENSE MUTATION”=SNP causes a change in        the encoded amino acid (i.e., a non-synonymous coding SNP);        “SILENT MUTATION”=SNP does not cause a change in the encoded        amino acid (i.e., a synonymous coding SNP); “STOP CODON        MUTATION”=SNP is located in a stop codon; “NONSENSE        MUTATION”=SNP creates or destroys a stop codon; “UTR 5”=SNP is        located in a 5′ UTR of a transcript; “UTR 3”=SNP is located in a        3′ UTR of a transcript; “PUTATIVE UTR 5”=SNP is located in a        putative 5′ UTR; “PUTATIVE UTR 3”=SNP is located in a putative        3′ UTR; “DONOR SPLICE SITE”=SNP is located in a donor splice        site (5′ intron boundary); “ACCEPTOR SPLICE SITE”=SNP is located        in an acceptor splice site (3′ intron boundary); “CODING        REGION”=SNP is located in a protein-coding region of the        transcript; “EXON”=SNP is located in an exon; “INTRON”=SNP is        located in an intron; “hmCS”=SNP is located in a human-mouse        conserved segment; “TFBS”=SNP is located in a transcription        factor binding site; “UNKNOWN”=SNP type is not defined;        “INTERGENIC”=SNP is intergenic, i.e., outside of any gene        boundary].    -   Protein coding information (Table 1 only), where relevant, in        the format of [protein SEQ ID NO:#, amino acid position, (amino        acid-1, codon1) (amino acid-2, codon2)]. The information in this        field includes SEQ ID NO of the encoded protein sequence,        position of the amino acid residue within the protein identified        by the SEQ ID NO that is encoded by the codon containing the        SNP, amino acids (represented by one-letter amino acid codes)        that are encoded by the alternative SNP alleles (in the case of        stop codons, “X” is used for the one-letter amino acid code),        and alternative codons containing the alternative SNP        nucleotides which encode the amino acid residues (thus, for        example, for missense mutation-type SNPs, at least two different        amino acids and at least two different codons are generally        indicated; for silent mutation-type SNPs, one amino acid and at        least two different codons are generally indicated, etc.). In        instances where the SNP is located outside of a protein-coding        region (e.g., in a UTR region), “None” is indicated following        the protein SEQ ID NO.

Description of Table 3

Table 3 provides sequences (SEQ ID NOS: 505-783) of oligonucleotidesthat have been synthesized and used in the laboratory to assay the SNPsdisclosed in Tables 4-8 during the course of association studies toverify the association of these SNPs with coronary heart disease (CHD).The experiments that were conducted using these primers are explained indetail in Examples 1 and 2, below.

Table 3 provides the following:

-   -   the column labeled “hCV” lists the Celera identifier hCV number        for each SNP marker.    -   the column labeled “Alleles” designates the two alternative        alleles at the SNP site identified by the hCV identification        number that are targeted by the allele-specific        oligonucleotides.    -   allele-specific oligonucleotides with their respective SEQ ID        numbers are shown in the next two columns, “Sequence A        (allele-specific primer)” and “Sequence B (allele-specific        primer).” These two primers were used in conjunction with a        common primer in each PCR assay to genotype DNA samples for each        SNP marker. Note that alleles may be presented in Table 3 based        on a different orientation (i.e., the reverse complement)        relative to how the same alleles are presented in Tables 1 and        2.    -   common oligonucleotides with their respective SEQ ID numbers are        shown in the column, “Sequence C (common primer).” Each common        primer was used in conjunction with the two allele-specific        primers to genotype DNA samples for each SNP marker.

All sequences are given in the 5′ to 3′ direction.

Description of Table 4

Table 4 provides results of statistical analyses for certain SNPsdisclosed in Tables 1 and 2 (SNPs can be cross-referenced between tablesbased on their hCV identification numbers), and the association of theseSNPs with CHD. The experiment that provided this data is explained indetail in Example 1, below.

The statistical results provided in Table 4 show that the association ofthese SNPs with MI is supported by P values<0.1 in an allelic orgenotypic association test, the latter based on dominant or recessivemodes of inheritance.

In Table 4, the column labeled “SNP Marker Identifier” presents each SNPas identified by its unique Celera hCV identification number. The column“Gene Symbol” presents the standard symbol for the gene containing theSNP; i.e., the symbol approved by the Human Genome Organization (HUGO)Gene Nomenclature Committee. In the case where a gene symbol is notknown or the SNP is found in an intergenic region, the word “none” ispresent. The column labeled “Risk Allele” presents a variant nucleotidefor each of the identified SNPs. The allele may be presented in Table 4as the reverse complement relative to how the same allele is presentedin Tables 1 and/or 2. “CCF” samples were obtained from patients at theCleveland Clinic Foundation Heart Center. “UCSF” samples were from theUniversity of California at San Francisco. The columns labeled “Design”present the coronary endpoints that a particular SNP is associated with,in the respective CCF or UCSF study. Reference is made to the Design Keybelow the table for explanation. For example, in Design A, SNPassociation with MI was found when cases with MI were compared tocontrols who did not have MI or any other cardiovascular disease (CVD).In Design B, younger MI cases were compared to older controls that didnot have any CVD. The numbers of cases and controls genotyped for eachassay are provided. The column labeled “Stratum” lists the subgroups ofindividuals from cases and controls in which MI association wasobserved. Reference is made to the Stratum Key below the table for anexplanation of symbols used. “All” indicates that the association wasobserved in all individuals tested, “M” or “FM” indicates theassociation was observed in males or females, “S+” or “S-” indicates anassociation with MI was observed in smokers or non-smokers,respectively. “BP+” indicates the effect was observed in patients withhypertension, etc. The column labeled “Mode” indicates the genetic modelunder which the P value for association was calculated. Under agenotypic analysis (described in examples below), when two copies of theSNP are required to see the observed effect, the mode is recessive, or“Rec.” When one or two copies of the SNP are required to see theassociation, the mode is dominant, or “Dom.” When the association isfound by simply comparing the frequency of the allele in the casepopulation to the control population, the mode is “Allelic.” The allelicmode closely approximates an additive model. The column labeled “P val”indicates the results of either the asymptotic chi-square test forgenotypic association (Rec or Dom), or the Fisher Exact test (Allelic)to determine if the qualitative phenotype is a function of the SNPgenotype. The column labeled “OR” (odds ratio) indicates anapproximation of the relative risk for an individual for the definedendpoint associated with the SNP. An OR of less than one indicates thatthe allele is protective for MI, and an OR greater than one indicatesthe allele increases the risk of MI.

Note that SNPs can be cross-referenced between the tables herein basedon their hCV identification numbers. However, four of the SNPs that areincluded in the tables may possess two different hCV identificationnumbers, as follows:

-   -   hCV15965459 and hCV22274416 are directed to identical SNP        markers.    -   hCV1801149 and hCV25473208 are directed to identical SNP        markers.    -   hCV3185278 and hCV28026155 are directed to identical SNP        markers.    -   hCV9626088 and hCV26809148 are directed to identical SNP        markers.

Description of Table 5

Table 5 provides the results of statistical analyses for certain SNPsdisclosed in Tables 1 and 2; namely, the association of SNP alleles witha risk for MI based on case-control studies. The experiment thatprovided this data is explained in detail in Example 1, below. Note thatSNPs can be cross-referenced between tables based on their hCVidentification numbers. The statistical results provided in Table 5 showthat the association of these SNPs with MI is supported by P values<0.05in allelic association tests. The data presented were obtained fromindividually genotyped samples from UCSF. Case samples were limited topatients that had a history of MI, while controls had no history of MI.

In Table 5, the column labeled “Gene Symbol” presents the standardsymbol for the gene containing the SNP; i.e., the symbol approved by theHuman Genome Organization (HUGO) Gene Nomenclature Committee. The columnlabeled “SNP Marker Identifier” presents each SNP as identified by itsunique Celera identifier, the hCV number. The column labeled “RiskAllele” presents the SNP allele for which the odds ratio was >1.0 forcases vs. controls. Each allele may be presented in Tables 1 and/or 2 asthe complement of the allele presented in Table 5; e.g., “G” may bepresented as its complement, “C.” The column labeled “P value” indicatesthe results of the Fisher Exact test, to determine the association ofone allele with risk for MI. The column labeled “OR” (odds ratio) showsan approximation of the relative MI risk for individuals with the riskallele, based on the observed frequencies of alleles in cases vs.controls. An OR less than one would indicate an allele is protective forMI, and an OR greater than one indicates the allele is associated withan increased risk of MI. Also shown is the 90% confidence intervalcalculated around each OR presented (“OR 90% CI”).

Description of Table 6

Table 6 provides the results of statistical analyses for certain SNPsdisclosed in Tables 1 and 2; namely, the association of SNP genotypeswith a risk for MI based on case-control studies. The experiment thatprovided this data is explained in detail in Example 1, below. Thestatistical results provided in Table 6 show that the association ofthese SNPs with MI is supported by P values<0.05 in genotypicassociation tests, when cases are heterozygous or homozygous for therisk allele, depending on the SNP. The data presented were obtained fromindividually genotyped samples from UCSF. Case samples were limited topatients that had a history of MI, while controls had no history of MI.The numbers of cases and controls of each genotype are provided in thistable. P values for genotypic association with MI were determined usingthe asymptotic chi-square test.

Description of Table 7

Table 7 provides the results of statistical analyses for certain SNPsdisclosed in Tables 1 and 2; namely, the association of SNP genotypeswith a risk for MI based on case-control studies. The SNPs of table 7were selected from genes in which other SNPs were found to be accociatedwith MI (Example 1 below, and Tables 5-6).

The statistical results provided in Table 7 show that the association ofthese SNPs with MI is supported by P values<0.10 in allelic or genotypicassociation tests, depending on the SNP. The data presented wereobtained from individually genotyped samples from UCSF. Case sampleswere limited to patients that had a history of MI, while controls had nohistory of MI. The column “Gene Symbol” indicates the gene region fromwhich the investigated markers were obtained. P values for genotypicassociation with MI (Dom or Rec) were determined using the asymptoticchi-square test; allelic association P value were obtained using theFisher Exact test.

Description of Table 8

Table 8 provides the results of a statistical analysis of SNPhCV3130332, disclosed in Tables 1 and 2; namely, the association thisSNP with a risk for stenosis based on case-control studies. Theexperiment that provided this data is explained in detail in Example 2,below.

The statistical results provided in Table 8 show that the association ofthis SNP with stenosis is supported by P values<0.05 in allelic andgenotypic (Dom/Rec) association tests. The data presented were obtainedfrom individually genotyped samples from CCF and UCSF. Case samples werelimited to patients with the most severe stenosis, while controls hadthe least severe stenosis and no MI history, as described in Example 2,below.

In Table 8, the column labeled “Gene Symbol” presents the standardsymbol for the gene containing the SNP. The column labeled “SNP MarkerIdentifier” presents the marker as identified by its unique Celeraidentifier, the hCV number. The column labeled “Risk Allele” presentsthe SNP allele for which the odds ratio was >1.0 for cases vs. controls.The columns labeled “P value” indicate the results (in CCF and UCSFsamples) of the Fisher Exact test to determine the association of oneallele with risk for MI, or the results of the asymptotic chi-squaretest, in the case of genotypic (dominant/recessive) association withstenosis. The columns labeled “OR” (odds ratio) show an approximation ofthe relative stenosis risk for individuals with the risk allele, basedon the observed frequencies of the risk allele in cases vs. controls. AnOR less than one would indicate an allele is protective for stenosis,and an OR greater than one indicates the allele is associated with anincreased risk of stenosis. Also shown are the 95% confidence intervalsfor the two sample sets, calculated around each OR presented (“OR 95%CI”).

Description of Table 9

Table 9 provides a list of the sample LD SNPs that are related to andderived from an interrogated SNP. These LD SNPs are provided as anexample of the groups of SNPs which can also serve as markers fordisease association based on their being in LD with the interrogatedSNP. The criteria and process of selecting such LD SNPs, including thecalculation of the r² value and the r² threshold value, are described inExample 3, below.

In Table 9, the column labeled “Interrogated SNP” presents each markeras identified by its unique identifier, the hCV number. The columnlabeled “Interrogated rs” presents the publicly known identifier rsnumber for the corresponding hCV number. The column labeled “LD SNP”presents the hCV numbers of the LD SNPs that are derived from theircorresponding interrogated SNPs. The column labeled “LD SNP rs” presentsthe publicly known rs number for the corresponding hCV number. Thecolumn labeled “Power” presents the level of power where the r²threshold is set. For example, when power is set at 70%, the thresholdr² value calculated therefrom is the minimum r² that an LD SNP must havein reference to an interrogated SNP, in order for the LD SNP to beclassified as a marker capable of being associated with a diseasephenotype at greater than 70% probability. The column labeled “Thresholdr²” presents the minimum value of r ² that an LD SNP must meet inreference to an interrogated SNP in order to qualify as an LD SNP. Thecolumn labeled “r²” presents the actual r² value of the LD SNP inreference to the interrogated SNP to which it is related.

DESCRIPTION OF THE FIGURE

FIG. 1 provides a diagrammatic representation of a computer-baseddiscovery system containing the SNP information of the present inventionin computer readable form.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides SNPs associated with CHD, and inparticular stenosis and MI, nucleic acid molecules containing SNPs,methods and reagents for the detection of the SNPs disclosed herein,uses of these SNPs for the development of detection reagents, and assaysor kits that utilize such reagents. The stenosis or MI-associated SNPsdisclosed herein are useful for diagnosing, screening for, andevaluating predisposition to stenosis or MI and related pathologies inhumans. Furthermore, such SNPs and their encoded products are usefultargets for the development of therapeutic agents.

A large number of SNPs have been identified from re-sequencing DNA from39 individuals, and they are indicated as “Applera” SNP source in Tables1-2. Their allele frequencies observed in each of the Caucasian andAfrican-American ethnic groups are provided. Additional SNPs includedherein were previously identified during shotgun sequencing and assemblyof the human genome, and they are indicated as “Celera” SNP source inTables 1-2. Furthermore, the information provided in Table 1-2,particularly the allele frequency information obtained from 39individuals and the identification of the precise position of each SNPwithin each gene/transcript, allows haplotypes (i.e., groups of SNPsthat are co-inherited) to be readily inferred. The present inventionencompasses SNP haplotypes, as well as individual SNPs.

Thus, the present invention provides individual SNPs associated withCHD, and in particular stenosis and MI, as well as combinations of SNPsand haplotypes in genetic regions associated with CHD, and in particularstenosis and MI, polymorphic/variant transcript sequences (SEQ ID NOS:1-67) and genomic sequences (SEQ ID NOS: 229-299) containing SNPs,encoded amino acid sequences (SEQ ID NOS: 68-134), and bothtranscript-based SNP context sequences (SEQ ID NOS: 135-228) andgenomic-based SNP context sequences (SEQ ID NOS: 300-504) (transcriptsequences, protein sequences, and transcript-based SNP context sequencesare provided in Table 1 and the Sequence Listing; genomic sequences andgenomic-based SNP context sequences are provided in Table 2 and theSequence Listing), methods of detecting these polymorphisms in a testsample, methods of determining the risk of an individual of having ordeveloping stenosis and/or MI, methods of screening for compounds usefulfor treating disorders associated with a variant gene/protein such asstenosis or MI, compounds identified by these screening methods, methodsof using the disclosed SNPs to select a treatment strategy, methods oftreating a disorder associated with a variant gene/protein (i.e.,therapeutic methods), methods of determining if an individual is likelyto respond to a specific treatment such as statins and methods of usingthe SNPs of the present invention for human identification.

The present invention provides novel SNPs associated with stenosisand/or MI, as well as SNPs that were previously known in the art, butwere not previously known to be associated with stenosis or MI.Accordingly, the present invention provides novel compositions andmethods based on the novel SNPs disclosed herein, and also providesnovel methods of using the known, but previously unassociated, SNPs inmethods relating to stenosis or MI (e.g., for diagnosing MI, etc.). InTables 1-2, known SNPs are identified based on the public database inwhich they have been observed, which is indicated as one or more of thefollowing SNP types: “dbSNP”=SNP observed in dbSNP, “HGBASE”=SNPobserved in HGBASE, and “HGMD”=SNP observed in the Human Gene MutationDatabase (HGMD). Novel SNPs for which the SNP source is only “Applera”and none other, i.e., those that have not been observed in any publicdatabases and which were also not observed during shotgun sequencing andassembly of the Celera human genome sequence (i.e., “Celera” SNPsource), are also noted in the tables.

Particular SNP alleles of the present invention can be associated witheither an increased risk of having or developing stenosis or MI, or adecreased risk of having or developing stenosis or MI. SNP alleles thatare associated with a decreased risk of having or developing stenosis orMI may be referred to as “protective” alleles, and SNP alleles that areassociated with an increased risk of having or developing stenosi or MImay be referred to as “susceptibility” alleles, “risk” alleles, or “riskfactors”. Thus, whereas certain SNPs (or their encoded products) can beassayed to determine whether an individual possesses a SNP allele thatis indicative of an increased risk of having or developing stenosis orMI (i.e., a susceptibility allele), other SNPs (or their encodedproducts) can be assayed to determine whether an individual possesses aSNP allele that is indicative of a decreased risk of having ordeveloping stenosis or MI (i.e., a protective allele). Similarly,particular SNP alleles of the present invention can be associated witheither an increased or decreased likelihood of responding to aparticular treatment or therapeutic compound, or an increased ordecreased likelihood of experiencing toxic effects from a particulartreatment or therapeutic compound. The term “altered” may be used hereinto encompass either of these two possibilities (e.g., an increased or adecreased risk/likelihood).

Those skilled in the art will readily recognize that nucleic acidmolecules may be double-stranded molecules and that reference to aparticular site on one strand refers, as well, to the corresponding siteon a complementary strand. In defining a SNP position, SNP allele, ornucleotide sequence, reference to an adenine, a thymine (uridine), acytosine, or a guanine at a particular site on one strand of a nucleicacid molecule also defines the thymine (uridine), adenine, guanine, orcytosine (respectively) at the corresponding site on a complementarystrand of the nucleic acid molecule. Thus, reference may be made toeither strand in order to refer to a particular SNP position, SNPallele, or nucleotide sequence. Probes and primers, may be designed tohybridize to either strand and SNP genotyping methods disclosed hereinmay generally target either strand. Throughout the specification, inidentifying a SNP position, reference is generally made to theprotein-encoding strand, only for the purpose of convenience.

References to variant peptides, polypeptides, or proteins of the presentinvention include peptides, polypeptides, proteins, or fragmentsthereof, that contain at least one amino acid residue that differs fromthe corresponding amino acid sequence of the art-knownpeptide/polypeptide/protein (the art-known protein may beinterchangeably referred to as the “wild-type,” “reference,” or “normal”protein). Such variant peptides/polypeptides/proteins can result from acodon change caused by a nonsynonymous nucleotide substitution at aprotein-coding SNP position (i.e., a missense mutation) disclosed by thepresent invention. Variant peptides/polypeptides/proteins of the presentinvention can also result from a nonsense mutation, i.e., a SNP thatcreates a premature stop codon, a SNP that generates a read-throughmutation by abolishing a stop codon, or due to any SNP disclosed by thepresent invention that otherwise alters the structure,function/activity, or expression of a protein, such as a SNP in aregulatory region (e.g. a promoter or enhancer) or a SNP that leads toalternative or defective splicing, such as a SNP in an intron or a SNPat an exon/intron boundary. As used herein, the terms “polypeptide,”“peptide,” and “protein” are used interchangeably.

Isolated Nucleic Acid Molecules and SNP Detection Reagents & Kits

Tables 1 and 2 provide a variety of information about each SNP of thepresent invention that is associated with CHD, and in particularstenosis and MI, including the transcript sequences (SEQ ID NOS: 1-67),genomic sequences (SEQ ID NOS: 229-299), and protein sequences (SEQ IDNOS: 68-134) of the encoded gene products (with the SNPs indicated byIUB codes in the nucleic acid sequences). In addition, Tables 1 and 2include SNP context sequences, which generally include 100 nucleotideupstream (5′) plus 100 nucleotides downstream (3′) of each SNP position(SEQ ID NOS: 135-228 correspond to transcript-based SNP contextsequences disclosed in Table 1, and SEQ ID NOS: 300-504 correspond togenomic-based context sequences disclosed in Table 2), the alternativenucleotides (alleles) at each SNP position, and additional informationabout the variant where relevant, such as SNP type (coding, missense,splice site, UTR, etc.), human populations in which the SNP wasobserved, observed allele frequencies, information about the encodedprotein, etc.

Isolated Nucleic Acid Molecules

The present invention provides isolated nucleic acid molecules thatcontain one or more SNPs disclosed Table 1 and/or Table 2. Isolatednucleic acid molecules containing one or more SNPs disclosed in at leastone of Tables 1-2 may be interchangeably referred to throughout thepresent text as “SNP-containing nucleic acid molecules”. Isolatednucleic acid molecules may optionally encode a full-length variantprotein or fragment thereof. The isolated nucleic acid molecules of thepresent invention also include probes and primers (which are describedin greater detail below in the section entitled “SNP DetectionReagents”), which may be used for assaying the disclosed SNPs, andisolated full-length genes, transcripts, cDNA molecules, and fragmentsthereof, which may be used for such purposes as expressing an encodedprotein.

As used herein, an “isolated nucleic acid molecule” generally is onethat contains a SNP of the present invention or one that hybridizes tosuch molecule such as a nucleic acid with a complementary sequence, andis separated from most other nucleic acids present in the natural sourceof the nucleic acid molecule. Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule containing a SNP of the presentinvention, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. A nucleicacid molecule can be fused to other coding or regulatory sequences andstill be considered “isolated”. Nucleic acid molecules present innon-human transgenic animals, which do not naturally occur in theanimal, are also considered “isolated”. For example, recombinant DNAmolecules contained in a vector are considered “isolated”. Furtherexamples of “isolated” DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells, and purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the isolated SNP-containing DNAmolecules of the present invention. Isolated nucleic acid moleculesaccording to the present invention further include such moleculesproduced synthetically.

Generally, an isolated SNP-containing nucleic acid molecule comprisesone or more SNP positions disclosed by the present invention withflanking nucleotide sequences on either side of the SNP positions. Aflanking sequence can include nucleotide residues that are naturallyassociated with the SNP site and/or heterologous nucleotide sequences.Preferably the flanking sequence is up to about 500, 300, 100, 60, 50,30, 25, 20, 15, 10, 8, or 4 nucleotides (or any other length in-between)on either side of a SNP position, or as long as the full-length gene orentire protein-coding sequence (or any portion thereof such as an exon),especially if the SNP-containing nucleic acid molecule is to be used toproduce a protein or protein fragment.

For full-length genes and entire protein-coding sequences, a SNPflanking sequence can be, for example, up to about 5 KB, 4 KB, 3 KB, 2KB, 1 KB on either side of the SNP. Furthermore, in such instances, theisolated nucleic acid molecule comprises exonic sequences (includingprotein-coding and/or non-coding exonic sequences), but may also includeintronic sequences. Thus, any protein coding sequence may be eithercontiguous or separated by introns. The important point is that thenucleic acid is isolated from remote and unimportant flanking sequencesand is of appropriate length such that it can be subjected to thespecific manipulations or uses described herein such as recombinantprotein expression, preparation of probes and primers for assaying theSNP position, and other uses specific to the SNP-containing nucleic acidsequences.

An isolated SNP-containing nucleic acid molecule can comprise, forexample, a full-length gene or transcript, such as a gene isolated fromgenomic DNA (e.g., by cloning or PCR amplification), a cDNA molecule, oran mRNA transcript molecule. Polymorphic transcript sequences arereferred to in Table 1 and provided in the Sequence Listing (SEQ ID NOS:1-67), and polymorphic genomic sequences are referred to in Table 2 andprovided in the Sequence Listing (SEQ ID NOS: 229-299). Furthermore,fragments of such full-length genes and transcripts that contain one ormore SNPs disclosed herein are also encompassed by the presentinvention, and such fragments may be used, for example, to express anypart of a protein, such as a particular functional domain or anantigenic epitope.

Thus, the present invention also encompasses fragments of the nucleicacid sequences as disclosed in Tables 1-2 (transcript sequences arereferred to in Table 1 as SEQ ID NOS: 1-67, genomic sequences arereferred to in Table 2 as SEQ ID NOS: 229-299, transcript-based SNPcontext sequences are referred to in Table 1 as SEQ ID NO: 135-228, andgenomic-based SNP context sequences are referred to in Table 2 as SEQ IDNO: 300-504) and their complements. The actual sequences referred to inthe tables are provided in the Sequence Listing. A fragment typicallycomprises a contiguous nucleotide sequence at least about 8 or morenucleotides, more preferably at least about 12 or more nucleotides, andeven more preferably at least about 16 or more nucleotides. Further, afragment could comprise at least about 18, 20, 22, 25, 30, 40, 50, 60,80, 100, 150, 200, 250 or 500 (or any other number in-between)nucleotides in length. The length of the fragment will be based on itsintended use. For example, the fragment can encode epitope-bearingregions of a variant peptide or regions of a variant peptide that differfrom the normal/wild-type protein, or can be useful as a polynucleotideprobe or primer. Such fragments can be isolated using the nucleotidesequences provided in Table 1 and/or Table 2 for the synthesis of apolynucleotide probe. A labeled probe can then be used, for example, toscreen a cDNA library, genomic DNA library, or mRNA to isolate nucleicacid corresponding to the coding region. Further, primers can be used inamplification reactions, such as for purposes of assaying one or moreSNPs sites or for cloning specific regions of a gene.

An isolated nucleic acid molecule of the present invention furtherencompasses a SNP-containing polynucleotide that is the product of anyone of a variety of nucleic acid amplification methods, which are usedto increase the copy numbers of a polynucleotide of interest in anucleic acid sample. Such amplification methods are well known in theart, and they include but are not limited to, polymerase chain reaction(PCR) (U.S. Pat. Nos. 4,683,195; and 4,683,202; PCR Technology:Principles and Applications for DNA Amplification, ed. H. A. Erlich,Freeman Press, NY, N.Y., 1992), ligase chain reaction (LCR) (Wu andWallace, Genomics 4:560, 1989; Landegren et al., Science 241:1077,1988), strand displacement amplification (SDA) (U.S. Pat. Nos.5,270,184; and 5,422,252), transcription-mediated amplification (TMA)(U.S. Pat. No. 5,399,491), linked linear amplification (LLA) (U.S. Pat.No. 6,027,923), and the like, and isothermal amplification methods suchas nucleic acid sequence based amplification (NASBA), and self-sustainedsequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874, 1990). Based on such methodologies, a person skilled in the artcan readily design primers in any suitable regions 5′ and 3′ to a SNPdisclosed herein. Such primers may be used to amplify DNA of any lengthso long that it contains the SNP of interest in its sequence.

As used herein, an “amplified polynucleotide” of the invention is aSNP-containing nucleic acid molecule whose amount has been increased atleast two fold by any nucleic acid amplification method performed invitro as compared to its starting amount in a test sample. In otherpreferred embodiments, an amplified polynucleotide is the result of atleast ten fold, fifty fold, one hundred fold, one thousand fold, or eventen thousand fold increase as compared to its starting amount in a testsample. In a typical PCR amplification, a polynucleotide of interest isoften amplified at least fifty thousand fold in amount over theunamplified genomic DNA, but the precise amount of amplification neededfor an assay depends on the sensitivity of the subsequent detectionmethod used.

Generally, an amplified polynucleotide is at least about 16 nucleotidesin length. More typically, an amplified polynucleotide is at least about20 nucleotides in length. In a preferred embodiment of the invention, anamplified polynucleotide is at least about 30 nucleotides in length. Ina more preferred embodiment of the invention, an amplifiedpolynucleotide is at least about 32, 40, 45, 50, or 60 nucleotides inlength. In yet another preferred embodiment of the invention, anamplified polynucleotide is at least about 100, 200, 300, 400, or 500nucleotides in length. While the total length of an amplifiedpolynucleotide of the invention can be as long as an exon, an intron orthe entire gene where the SNP of interest resides, an amplified productis typically up to about 1,000 nucleotides in length (although certainamplification methods may generate amplified products greater than 1000nucleotides in length). More preferably, an amplified polynucleotide isnot greater than about 600-700 nucleotides in length. It is understoodthat irrespective of the length of an amplified polynucleotide, a SNP ofinterest may be located anywhere along its sequence.

In a specific embodiment of the invention, the amplified product is atleast about 201 nucleotides in length, comprises one of thetranscript-based context sequences or the genomic-based contextsequences shown in Tables 1-2. Such a product may have additionalsequences on its 5′ end or 3′ end or both. In another embodiment, theamplified product is about 101 nucleotides in length, and it contains aSNP disclosed herein. Preferably, the SNP is located at the middle ofthe amplified product (e.g. at position 101 in an amplified product thatis 201 nucleotides in length, or at position 51 in an amplified productthat is 101 nucleotides in length), or within 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 15, or 20 nucleotides from the middle of the amplified product(however, as indicated above, the SNP of interest may be locatedanywhere along the length of the amplified product).

The present invention provides isolated nucleic acid molecules thatcomprise, consist of, or consist essentially of one or morepolynucleotide sequences that contain one or more SNPs disclosed herein,complements thereof, and SNP-containing fragments thereof.

Accordingly, the present invention provides nucleic acid molecules thatconsist of any of the nucleotide sequences shown in Table 1 and/or Table2 (transcript sequences are referred to in Table 1 as SEQ ID NOS: 1-67,genomic sequences are referred to in Table 2 as SEQ ID NOS: 229-299,transcript-based SNP context sequences are referred to in Table 1 as SEQID NO: 135-228, and genomic-based SNP context sequences are referred toin Table 2 as SEQ ID NO: 300-504), or any nucleic acid molecule thatencodes any of the variant proteins referred to in Table 1 (SEQ ID NOS:68-134). The actual sequences referred to in the tables are provided inthe Sequence Listing. A nucleic acid molecule consists of a nucleotidesequence when the nucleotide sequence is the complete nucleotidesequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of any of the nucleotide sequences referred to inTable 1 and/or Table 2 (transcript sequences are referred to in Table 1as SEQ ID NOS:1-67, genomic sequences are referred to in Table 2 as SEQID NOS: 229-299, transcript-based SNP context sequences are referred toin Table 1 as SEQ ID NO: 135-228, and genomic-based SNP contextsequences are referred to in Table 2 as SEQ ID NO: 300-504), or anynucleic acid molecule that encodes any of the variant proteins referredto in Table 1 (SEQ ID NOS: 68-134). The actual sequences referred to inthe tables are provided in the Sequence Listing. A nucleic acid moleculeconsists essentially of a nucleotide sequence when such a nucleotidesequence is present with only a few additional nucleotide residues inthe final nucleic acid molecule.

The present invention further provides nucleic acid molecules thatcomprise any of the nucleotide sequences shown in Table 1 and/or Table 2or a SNP-containing fragment thereof (transcript sequences are referredto in Table 1 as SEQ ID NOS: 1-67, genomic sequences are referred to inTable 2 as SEQ ID NOS: 229-299, transcript-based SNP context sequencesare referred to in Table 1 as SEQ ID NO: 135-228, and genomic-based SNPcontext sequences are referred to in Table 2 as SEQ ID NO: 300-504), orany nucleic acid molecule that encodes any of the variant proteinsprovided in Table 1 (SEQ ID NOS: 147-292). The actual sequences referredto in the tables are provided in the Sequence Listing. A nucleic acidmolecule comprises a nucleotide sequence when the nucleotide sequence isat least part of the final nucleotide sequence of the nucleic acidmolecule. In such a fashion, the nucleic acid molecule can be only thenucleotide sequence or have additional nucleotide residues, such asresidues that are naturally associated with it or heterologousnucleotide sequences. Such a nucleic acid molecule can have one to a fewadditional nucleotides or can comprise many more additional nucleotides.A brief description of how various types of these nucleic acid moleculescan be readily made and isolated is provided below, and such techniquesare well known to those of ordinary skill in the art (Sambrook andRussell, 2000, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, NY).

The isolated nucleic acid molecules can encode mature proteins plusadditional amino or carboxyl-terminal amino acids or both, or aminoacids interior to the mature peptide (when the mature form has more thanone peptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life, or facilitatemanipulation of a protein for assay or production. As generally is thecase in situ, the additional amino acids may be processed away from themature protein by cellular enzymes.

Thus, the isolated nucleic acid molecules include, but are not limitedto, nucleic acid molecules having a sequence encoding a peptide alone, asequence encoding a mature peptide and additional coding sequences suchas a leader or secretory sequence (e.g., a pre-pro or pro-proteinsequence), a sequence encoding a mature peptide with or withoutadditional coding sequences, plus additional non-coding sequences, forexample introns and non-coding 5′ and 3′ sequences such as transcribedbut untranslated sequences that play a role in, for example,transcription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding, and/or stability of mRNA. In addition, thenucleic acid molecules may be fused to heterologous marker sequencesencoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA, which may beobtained, for example, by molecular cloning or produced by chemicalsynthetic techniques or by a combination thereof (Sambrook and Russell,2000, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,NY). Furthermore, isolated nucleic acid molecules, particularly SNPdetection reagents such as probes and primers, can also be partially orcompletely in the form of one or more types of nucleic acid analogs,such as peptide nucleic acid (PNA) (U.S. Pat. Nos. 5,539,082; 5,527,675;5,623,049; 5,714,331). The nucleic acid, especially DNA, can bedouble-stranded or single-stranded. Single-stranded nucleic acid can bethe coding strand (sense strand) or the complementary non-coding strand(anti-sense strand). DNA, RNA, or PNA segments can be assembled, forexample, from fragments of the human genome (in the case of DNA or RNA)or single nucleotides, short oligonucleotide linkers, or from a seriesof oligonucleotides, to provide a synthetic nucleic acid molecule.Nucleic acid molecules can be readily synthesized using the sequencesprovided herein as a reference; oligonucleotide and PNA oligomersynthesis techniques are well known in the art (see, e.g., Corey,“Peptide nucleic acids: expanding the scope of nucleic acidrecognition”, Trends Biotechnol. 1997 June; 15(6):224-9, and Hyrup etal., “Peptide nucleic acids (PNA): synthesis, properties and potentialapplications”, Bioorg Med. Chem. 1996 January; 4(1):5-23). Furthermore,large-scale automated oligonucleotide/PNA synthesis (including synthesison an array or bead surface or other solid support) can readily beaccomplished using commercially available nucleic acid synthesizers,such as the Applied Biosystems (Foster City, Calif.) 3900High-Throughput DNA Synthesizer or Expedite 8909 Nucleic Acid SynthesisSystem, and the sequence information provided herein.

The present invention encompasses nucleic acid analogs that containmodified, synthetic, or non-naturally occurring nucleotides orstructural elements or other alternative/modified nucleic acidchemistries known in the art. Such nucleic acid analogs are useful, forexample, as detection reagents (e.g., primers/probes) for detecting oneor more SNPs identified in Table 1 and/or Table 2. Furthermore,kits/systems (such as beads, arrays, etc.) that include these analogsare also encompassed by the present invention. For example, PNAoligomers that are based on the polymorphic sequences of the presentinvention are specifically contemplated. PNA oligomers are analogs ofDNA in which the phosphate backbone is replaced with a peptide-likebackbone (Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters,4: 1081-1082 (1994), Petersen et al., Bioorganic & Medicinal ChemistryLetters, 6: 793-796 (1996), Kumar et al., Organic Letters 3(9):1269-1272 (2001), WO96/04000). PNA hybridizes to complementary RNA orDNA with higher affinity and specificity than conventionaloligonucleotides and oligonucleotide analogs. The properties of PNAenable novel molecular biology and biochemistry applicationsunachievable with traditional oligonucleotides and peptides.

Additional examples of nucleic acid modifications that improve thebinding properties and/or stability of a nucleic acid include the use ofbase analogs such as inosine, intercalators (U.S. Pat. No. 4,835,263)and the minor groove binders (U.S. Pat. No. 5,801,115). Thus, referencesherein to nucleic acid molecules, SNP-containing nucleic acid molecules,SNP detection reagents (e.g., probes and primers),oligonucleotides/polynucleotides include PNA oligomers and other nucleicacid analogs. Other examples of nucleic acid analogs andalternative/modified nucleic acid chemistries known in the art aredescribed in Current Protocols in Nucleic Acid Chemistry, John Wiley &Sons, N.Y. (2002).

The present invention further provides nucleic acid molecules thatencode fragments of the variant polypeptides disclosed herein as well asnucleic acid molecules that encode obvious variants of such variantpolypeptides. Such nucleic acid molecules may be naturally occurring,such as paralogs (different locus) and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Non-naturally occurring variants may be made by mutagenesistechniques, including those applied to nucleic acid molecules, cells, ororganisms. Accordingly, the variants can contain nucleotidesubstitutions, deletions, inversions and insertions (in addition to theSNPs disclosed in Tables 1-2). Variation can occur in either or both thecoding and non-coding regions. The variations can produce conservativeand/or non-conservative amino acid substitutions.

Further variants of the nucleic acid molecules disclosed in Tables 1-2,such as naturally occurring allelic variants (as well as orthologs andparalogs) and synthetic variants produced by mutagenesis techniques, canbe identified and/or produced using methods well known in the art. Suchfurther variants can comprise a nucleotide sequence that shares at least70-80%, 80-85%, 85-90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity with a nucleic acid sequence disclosed in Table 1and/or Table 2 (or a fragment thereof) and that includes a novel SNPallele disclosed in Table 1 and/or Table 2. Further, variants cancomprise a nucleotide sequence that encodes a polypeptide that shares atleast 70-80%, 80-85%, 85-90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity with a polypeptide sequence disclosed in Table 1(or a fragment thereof) and that includes a novel SNP allele disclosedin Table 1 and/or Table 2. Thus, an aspect of the present invention thatis specifically contemplated are isolated nucleic acid molecules thathave a certain degree of sequence variation compared with the sequencesshown in Tables 1-2, but that contain a novel SNP allele disclosedherein. In other words, as long as an isolated nucleic acid moleculecontains a novel SNP allele disclosed herein, other portions of thenucleic acid molecule that flank the novel SNP allele can vary to somedegree from the specific transcript, genomic, and context sequencesreferred to and shown in Tables 1-2, and can encode a polypeptide thatvaries to some degree from the specific polypeptide sequences referredto in Table 1.

To determine the percent identity of two amino acid sequences or twonucleotide sequences of two molecules that share sequence homology, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In a preferred embodiment, atleast 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of areference sequence is aligned for comparison purposes. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein, amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991). In a preferred embodiment, the percent identity between two aminoacid sequences is determined using the Needleman and Wunsch algorithm(J. Mol. Biol. (48):444-453 (1970)) which has been incorporated into theGAP program in the GCG software package, using either a Blossom 62matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or4 and a length weight of 1, 2, 3, 4, 5, or 6.

In yet another preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (Devereux, J., et al., Nucleic Acids Res. 12(1):387(1984)), using a NWSgapdna CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In anotherembodiment, the percent identity between two amino acid or nucleotidesequences is determined using the algorithm of E. Myers and W. Miller(CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4.

The nucleotide and amino acid sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNTBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. In addition to BLAST, examples of othersearch and sequence comparison programs used in the art include, but arenot limited to, FASTA (Pearson, Methods Mol. Biol. 25, 365-389 (1994))and KERR (Dufresne et al., Nat Biotechnol 2002 December;20(12):1269-71). For further information regarding bioinformaticstechniques, see Current Protocols in Bioinformatics, John Wiley & Sons,Inc., N.Y.

The present invention further provides non-coding fragments of thenucleic acid molecules disclosed in Table 1 and/or Table 2. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, intronic sequences, 5′ untranslatedregions (UTRs), 3′ untranslated regions, gene modulating sequences andgene termination sequences. Such fragments are useful, for example, incontrolling heterologous gene expression and in developing screens toidentify gene-modulating agents.

SNP Detection Reagents In a specific aspect of the present invention,the SNPs disclosed in Table 1 and/or Table 2, and their associatedtranscript sequences (referred to in Table 1 as SEQ ID NOS: 1-67),genomic sequences (referred to in Table 2 as SEQ ID NOS: 229-299), andcontext sequences (transcript-based context sequences are referred to inTable 1 as SEQ ID NOS: 135-228; genomic-based context sequences areprovided in Table 2 as SEQ ID NOS: 300-504), can be used for the designof SNP detection reagents. The actual sequences referred to in thetables are provided in the Sequence Listing. As used herein, a “SNPdetection reagent” is a reagent that specifically detects a specifictarget SNP position disclosed herein, and that is preferably specificfor a particular nucleotide (allele) of the target SNP position (i.e.,the detection reagent preferably can differentiate between differentalternative nucleotides at a target SNP position, thereby allowing theidentity of the nucleotide present at the target SNP position to bedetermined). Typically, such detection reagent hybridizes to a targetSNP-containing nucleic acid molecule by complementary base-pairing in asequence specific manner, and discriminates the target variant sequencefrom other nucleic acid sequences such as an art-known form in a testsample. An example of a detection reagent is a probe that hybridizes toa target nucleic acid containing one or more of the SNPs referred to inTable 1 and/or Table 2. In a preferred embodiment, such a probe candifferentiate between nucleic acids having a particular nucleotide(allele) at a target SNP position from other nucleic acids that have adifferent nucleotide at the same target SNP position. In addition, adetection reagent may hybridize to a specific region 5′ and/or 3′ to aSNP position, particularly a region corresponding to the contextsequences referred to in Table 1 and/or Table 2 (transcript-basedcontext sequences are referred to in Table 1 as SEQ ID NOS: 135-228;genomic-based context sequences are referred to in Table 2 as SEQ IDNOS: 300-504). Another example of a detection reagent is a primer thatacts as an initiation point of nucleotide extension along acomplementary strand of a target polynucleotide. The SNP sequenceinformation provided herein is also useful for designing primers, e.g.allele-specific primers, to amplify (e.g., using PCR) any SNP of thepresent invention.

In one preferred embodiment of the invention, a SNP detection reagent isan isolated or synthetic DNA or RNA polynucleotide probe or primer orPNA oligomer, or a combination of DNA, RNA and/or PNA, that hybridizesto a segment of a target nucleic acid molecule containing a SNPidentified in Table 1 and/or Table 2. A detection reagent in the form ofa polynucleotide may optionally contain modified base analogs,intercalators or minor groove binders. Multiple detection reagents suchas probes may be, for example, affixed to a solid support (e.g., arraysor beads) or supplied in solution (e.g., probe/primer sets for enzymaticreactions such as PCR, RT-PCR, TaqMan assays, or primer-extensionreactions) to form a SNP detection kit.

A probe or primer typically is a substantially purified oligonucleotideor PNA oligomer. Such oligonucleotide typically comprises a region ofcomplementary nucleotide sequence that hybridizes under stringentconditions to at least about 8, 10, 12, 16, 18, 20, 22, 25, 30, 40, 50,55, 60, 65, 70, 80, 90, 100, 120 (or any other number in-between) ormore consecutive nucleotides in a target nucleic acid molecule.Depending on the particular assay, the consecutive nucleotides caneither include the target SNP position, or be a specific region in closeenough proximity 5′ and/or 3′ to the SNP position to carry out thedesired assay.

Other preferred primer and probe sequences can readily be determinedusing the transcript sequences (SEQ ID NOS: 1-67), genomic sequences(SEQ ID NOS: 229-299), and SNP context sequences (transcript-basedcontext sequences are referred to in Table 1 as SEQ ID NOS: 135-228;genomic-based context sequences are referred to in Table 2 as SEQ IDNOS: 300-504) disclosed in the Sequence Listing and in Tables 1-2. Theactual sequences referred to in the tables are provided in the SequenceListing. It will be apparent to one of skill in the art that suchprimers and probes are directly useful as reagents for genotyping theSNPs of the present invention, and can be incorporated into anykit/system format.

In order to produce a probe or primer specific for a targetSNP-containing sequence, the gene/transcript and/or context sequencesurrounding the SNP of interest is typically examined using a computeralgorithm that starts at the 5′ or at the 3′ end of the nucleotidesequence. Typical algorithms will then identify oligomers of definedlength that are unique to the gene/SNP context sequence, have a GCcontent within a range suitable for hybridization, lack predictedsecondary structure that may interfere with hybridization, and/orpossess other desired characteristics or that lack other undesiredcharacteristics.

A primer or probe of the present invention is typically at least about 8nucleotides in length. In one embodiment of the invention, a primer or aprobe is at least about 10 nucleotides in length. In a preferredembodiment, a primer or a probe is at least about 12 nucleotides inlength. In a more preferred embodiment, a primer or probe is at leastabout 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.While the maximal length of a probe can be as long as the targetsequence to be detected, depending on the type of assay in which it isemployed, it is typically less than about 50, 60, 65, or 70 nucleotidesin length. In the case of a primer, it is typically less than about 30nucleotides in length. In a specific preferred embodiment of theinvention, a primer or a probe is within the length of about 18 andabout 28 nucleotides. However, in other embodiments, such as nucleicacid arrays and other embodiments in which probes are affixed to asubstrate, the probes can be longer, such as on the order of 30-70, 75,80, 90, 100, or more nucleotides in length (see the section belowentitled “SNP Detection Kits and Systems”).

For analyzing SNPs, it may be appropriate to use oligonucleotidesspecific for alternative SNP alleles. Such oligonucleotides that detectsingle nucleotide variations in target sequences may be referred to bysuch terms as “allele-specific oligonucleotides”, “allele-specificprobes”, or “allele-specific primers”. The design and use ofallele-specific probes for analyzing polymorphisms is described in,e.g., Mutation Detection A Practical Approach, ed. Cotton et al. OxfordUniversity Press, 1998; Saiki et al, Nature 324, 163-166 (1986);Dattagupta, EP235,726; and Saiki, WO 89/11548.

While the design of each allele-specific primer or probe depends onvariables such as the precise composition of the nucleotide sequencesflanking a SNP position in a target nucleic acid molecule, and thelength of the primer or probe, another factor in the use of primers andprobes is the stringency of the condition under which the hybridizationbetween the probe or primer and the target sequence is performed. Higherstringency conditions utilize buffers with lower ionic strength and/or ahigher reaction temperature, and tend to require a more perfect matchbetween probe/primer and a target sequence in order to form a stableduplex. If the stringency is too high, however, hybridization may notoccur at all. In contrast, lower stringency conditions utilize bufferswith higher ionic strength and/or a lower reaction temperature, andpermit the formation of stable duplexes with more mismatched basesbetween a probe/primer and a target sequence. By way of example and notlimitation, exemplary conditions for high stringency hybridizationconditions using an allele-specific probe are as follows:prehybridization with a solution containing 5× standard saline phosphateEDTA (SSPE), 0.5% NaDodSO₄ (SDS) at 55° C., and incubating probe withtarget nucleic acid molecules in the same solution at the sametemperature, followed by washing with a solution containing 2×SSPE, and0.1% SDS at 55° C. or room temperature.

Moderate stringency hybridization conditions may be used forallele-specific primer extension reactions with a solution containing,e.g., about 50 mM KCl at about 46° C. Alternatively, the reaction may becarried out at an elevated temperature such as 60° C. In anotherembodiment, a moderately stringent hybridization condition suitable foroligonucleotide ligation assay (OLA) reactions wherein two probes areligated if they are completely complementary to the target sequence mayutilize a solution of about 100 mM KCl at a temperature of 46° C.

In a hybridization-based assay, allele-specific probes can be designedthat hybridize to a segment of target DNA from one individual but do nothybridize to the corresponding segment from another individual due tothe presence of different polymorphic forms (e.g., alternative SNPalleles/nucleotides) in the respective DNA segments from the twoindividuals. Hybridization conditions should be sufficiently stringentthat there is a significant detectable difference in hybridizationintensity between alleles, and preferably an essentially binaryresponse, whereby a probe hybridizes to only one of the alleles orsignificantly more strongly to one allele. While a probe may be designedto hybridize to a target sequence that contains a SNP site such that theSNP site aligns anywhere along the sequence of the probe, the probe ispreferably designed to hybridize to a segment of the target sequencesuch that the SNP site aligns with a central position of the probe(e.g., a position within the probe that is at least three nucleotidesfrom either end of the probe). This design of probe generally achievesgood discrimination in hybridization between different allelic forms.

In another embodiment, a probe or primer may be designed to hybridize toa segment of target DNA such that the SNP aligns with either the 5′ mostend or the 3′ most end of the probe or primer. In a specific preferredembodiment that is particularly suitable for use in a oligonucleotideligation assay (U.S. Pat. No. 4,988,617), the 3′ most nucleotide of theprobe aligns with the SNP position in the target sequence.

Oligonucleotide probes and primers may be prepared by methods well knownin the art.

Chemical synthetic methods include, but are limited to, thephosphotriester method described by Narang et al., 1979, Methods inEnzymology 68:90; the phosphodiester method described by Brown et al.,1979, Methods in Enzymology 68:109, the diethylphosphoamidate methoddescribed by Beaucage et al., 1981, Tetrahedron Letters 22:1859; and thesolid support method described in U.S. Pat. No. 4,458,066.

Allele-specific probes are often used in pairs (or, less commonly, insets of 3 or 4, such as if a SNP position is known to have 3 or 4alleles, respectively, or to assay both strands of a nucleic acidmolecule for a target SNP allele), and such pairs may be identicalexcept for a one nucleotide mismatch that represents the allelicvariants at the SNP position. Commonly, one member of a pair perfectlymatches a reference form of a target sequence that has a more common SNPallele (i.e., the allele that is more frequent in the target population)and the other member of the pair perfectly matches a form of the targetsequence that has a less common SNP allele (i.e., the allele that israrer in the target population). In the case of an array, multiple pairsof probes can be immobilized on the same support for simultaneousanalysis of multiple different polymorphisms.

In one type of PCR-based assay, an allele-specific primer hybridizes toa region on a target nucleic acid molecule that overlaps a SNP positionand only primes amplification of an allelic form to which the primerexhibits perfect complementarity (Gibbs, 1989, Nucleic Acid Res. 172427-2448). Typically, the primer's 3′-most nucleotide is aligned withand complementary to the SNP position of the target nucleic acidmolecule. This primer is used in conjunction with a second primer thathybridizes at a distal site. Amplification proceeds from the twoprimers, producing a detectable product that indicates which allelicform is present in the test sample. A control is usually performed witha second pair of primers, one of which shows a single base mismatch atthe polymorphic site and the other of which exhibits perfectcomplementarity to a distal site. The single-base mismatch preventsamplification or substantially reduces amplification efficiency, so thateither no detectable product is formed or it is formed in lower amountsor at a slower pace. The method generally works most effectively whenthe mismatch is at the 3′-most position of the oligonucleotide (i.e.,the 3′-most position of the oligonucleotide aligns with the target SNPposition) because this position is most destabilizing to elongation fromthe primer (see, e.g., WO 93/22456). This PCR-based assay can beutilized as part of the TaqMan assay, described below.

In a specific embodiment of the invention, a primer of the inventioncontains a sequence substantially complementary to a segment of a targetSNP-containing nucleic acid molecule except that the primer has amismatched nucleotide in one of the three nucleotide positions at the3′-most end of the primer, such that the mismatched nucleotide does notbase pair with a particular allele at the SNP site. In a preferredembodiment, the mismatched nucleotide in the primer is the second fromthe last nucleotide at the 3′-most position of the primer. In a morepreferred embodiment, the mismatched nucleotide in the primer is thelast nucleotide at the 3′-most position of the primer.

In another embodiment of the invention, a SNP detection reagent of theinvention is labeled with a fluorogenic reporter dye that emits adetectable signal. While the preferred reporter dye is a fluorescentdye, any reporter dye that can be attached to a detection reagent suchas an oligonucleotide probe or primer is suitable for use in theinvention. Such dyes include, but are not limited to, Acridine, AMCA,BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Dabcyl, Edans, Eosin,Erythrosin, Fluorescein, 6-Fam, Tet, Joe, Hex, Oregon Green, Rhodamine,Rhodol Green, Tamra, Rox, and Texas Red.

In yet another embodiment of the invention, the detection reagent may befurther labeled with a quencher dye such as Tamra, especially when thereagent is used as a self-quenching probe such as a TaqMan (U.S. Pat.Nos. 5,210,015 and 5,538,848) or Molecular Beacon probe (U.S. Pat. Nos.5,118,801 and 5,312,728), or other stemless or linear beacon probe(Livak et al., 1995, PCR Method Appl. 4:357-362; Tyagi et al., 1996,Nature Biotechnology 14: 303-308; Nazarenko et al., 1997, Nucl. AcidsRes. 25:2516-2521; U.S. Pat. Nos. 5,866,336 and 6,117,635).

The detection reagents of the invention may also contain other labels,including but not limited to, biotin for streptavidin binding, haptenfor antibody binding, and oligonucleotide for binding to anothercomplementary oligonucleotide such as pairs of zipcodes.

The present invention also contemplates reagents that do not contain (orthat are complementary to) a SNP nucleotide identified herein but thatare used to assay one or more SNPs disclosed herein. For example,primers that flank, but do not hybridize directly to a target SNPposition provided herein are useful in primer extension reactions inwhich the primers hybridize to a region adjacent to the target SNPposition (i.e., within one or more nucleotides from the target SNPsite). During the primer extension reaction, a primer is typically notable to extend past a target SNP site if a particular nucleotide(allele) is present at that target SNP site, and the primer extensionproduct can be detected in order to determine which SNP allele ispresent at the target SNP site. For example, particular ddNTPs aretypically used in the primer extension reaction to terminate primerextension once a ddNTP is incorporated into the extension product (aprimer extension product which includes a ddNTP at the 3′-most end ofthe primer extension product, and in which the ddNTP is a nucleotide ofa SNP disclosed herein, is a composition that is specificallycontemplated by the present invention). Thus, reagents that bind to anucleic acid molecule in a region adjacent to a SNP site and that areused for assaying the SNP site, even though the bound sequences do notnecessarily include the SNP site itself, are also contemplated by thepresent invention.

SNP Detection Kits and Systems

A person skilled in the art will recognize that, based on the SNP andassociated sequence information disclosed herein, detection reagents canbe developed and used to assay any SNP of the present inventionindividually or in combination, and such detection reagents can bereadily incorporated into one of the established kit or system formatswhich are well known in the art. The terms “kits” and “systems”, as usedherein in the context of SNP detection reagents, are intended to referto such things as combinations of multiple SNP detection reagents, orone or more SNP detection reagents in combination with one or more othertypes of elements or components (e.g., other types of biochemicalreagents, containers, packages such as packaging intended for commercialsale, substrates to which SNP detection reagents are attached,electronic hardware components, etc.). Accordingly, the presentinvention further provides SNP detection kits and systems, including butnot limited to, packaged probe and primer sets (e.g., TaqManprobe/primer sets), arrays/microarrays of nucleic acid molecules, andbeads that contain one or more probes, primers, or other detectionreagents for detecting one or more SNPs of the present invention. Thekits/systems can optionally include various electronic hardwarecomponents; for example, arrays (“DNA chips”) and microfluidic systems(“lab-on-a-chip” systems) provided by various manufacturers typicallycomprise hardware components. Other kits/systems (e.g., probe/primersets) may not include electronic hardware components, but may becomprised of, for example, one or more SNP detection reagents (alongwith, optionally, other biochemical reagents) packaged in one or morecontainers.

In some embodiments, a SNP detection kit typically contains one or moredetection reagents and other components (e.g., a buffer, enzymes such asDNA polymerases or ligases, chain extension nucleotides such asdeoxynucleotide triphosphates, and in the case of Sanger-type DNAsequencing reactions, chain terminating nucleotides, positive controlsequences, negative control sequences, and the like) necessary to carryout an assay or reaction, such as amplification and/or detection of aSNP-containing nucleic acid molecule. A kit may further contain meansfor determining the amount of a target nucleic acid, and means forcomparing the amount with a standard, and can comprise instructions forusing the kit to detect the SNP-containing nucleic acid molecule ofinterest. In one embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out one or more assays todetect one or more SNPs disclosed herein. In a preferred embodiment ofthe present invention, SNP detection kits/systems are in the form ofnucleic acid arrays, or compartmentalized kits, includingmicrofluidic/lab-on-a-chip systems.

SNP detection kits/systems may contain, for example, one or more probes,or pairs of probes, that hybridize to a nucleic acid molecule at or neareach target SNP position. Multiple pairs of allele-specific probes maybe included in the kit/system to simultaneously assay large numbers ofSNPs, at least one of which is a SNP of the present invention. In somekits/systems, the allele-specific probes are immobilized to a substratesuch as an array or bead. For example, the same substrate can compriseallele-specific probes for detecting at least 1; 10; 100; 1000; 10,000;100,000 (or any other number in-between) or substantially all of theSNPs shown in Table 1 and/or Table 2.

The terms “arrays,” “microarrays,” and “DNA chips” are used hereininterchangeably to refer to an array of distinct polynucleotides affixedto a substrate, such as glass, plastic, paper, nylon or other type ofmembrane, filter, chip, or any other suitable solid support. Thepolynucleotides can be synthesized directly on the substrate, orsynthesized separate from the substrate and then affixed to thesubstrate. In one embodiment, the microarray is prepared and usedaccording to the methods described in U.S. Pat. No. 5,837,832, Chee etal., PCT application W095/11995 (Chee et al.), Lockhart, D. J. et al.(1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc.Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated hereinin their entirety by reference. In other embodiments, such arrays areproduced by the methods described by Brown et al., U.S. Pat. No.5,807,522.

Nucleic acid arrays are reviewed in the following references: Zammatteoet al., “New chips for molecular biology and diagnostics”, BiotechnolAnnu Rev. 2002; 8:85-101; Sosnowski et al., “Active microelectronicarray system for DNA hybridization, genotyping and pharmacogenomicapplications”, Psychiatr Genet. 2002 December; 12(4):181-92; Heller,“DNA microarray technology: devices, systems, and applications”, AnnuRev Biomed Eng. 2002; 4:129-53. Epub 2002 Mar. 22; Kolchinsky et al.,“Analysis of SNPs and other genomic variations using gel-based chips”,Hum Mutat. 2002 April; 19(4):343-60; and McGall et al., “High-densitygenechip oligonucleotide probe arrays”, Adv Biochem Eng Biotechnol.2002; 77:21-42.

Any number of probes, such as allele-specific probes, may be implementedin an array, and each probe or pair of probes can hybridize to adifferent SNP position. In the case of polynucleotide probes, they canbe synthesized at designated areas (or synthesized separately and thenaffixed to designated areas) on a substrate using a light-directedchemical process. Each DNA chip can contain, for example, thousands tomillions of individual synthetic polynucleotide probes arranged in agrid-like pattern and miniaturized (e.g., to the size of a dime).Preferably, probes are attached to a solid support in an ordered,addressable array.

A microarray can be composed of a large number of unique,single-stranded polynucleotides, usually either synthetic antisensepolynucleotides or fragments of cDNAs, fixed to a solid support. Typicalpolynucleotides are preferably about 6-60 nucleotides in length, morepreferably about 15-30 nucleotides in length, and most preferably about18-25 nucleotides in length. For certain types of microarrays or otherdetection kits/systems, it may be preferable to use oligonucleotidesthat are only about 7-20 nucleotides in length. In other types ofarrays, such as arrays used in conjunction with chemiluminescentdetection technology, preferred probe lengths can be, for example, about15-80 nucleotides in length, preferably about 50-70 nucleotides inlength, more preferably about 55-65 nucleotides in length, and mostpreferably about 60 nucleotides in length. The microarray or detectionkit can contain polynucleotides that cover the known 5′ or 3′ sequenceof a gene/transcript or target SNP site, sequential polynucleotides thatcover the full-length sequence of a gene/transcript; or uniquepolynucleotides selected from particular areas along the length of atarget gene/transcript sequence, particularly areas corresponding to oneor more SNPs disclosed in Table 1 and/or Table 2. Polynucleotides usedin the microarray or detection kit can be specific to a SNP or SNPs ofinterest (e.g., specific to a particular SNP allele at a target SNPsite, or specific to particular SNP alleles at multiple different SNPsites), or specific to a polymorphic gene/transcript orgenes/transcripts of interest.

Hybridization assays based on polynucleotide arrays rely on thedifferences in hybridization stability of the probes to perfectlymatched and mismatched target sequence variants. For SNP genotyping, itis generally preferable that stringency conditions used in hybridizationassays are high enough such that nucleic acid molecules that differ fromone another at as little as a single SNP position can be differentiated(e.g., typical SNP hybridization assays are designed so thathybridization will occur only if one particular nucleotide is present ata SNP position, but will not occur if an alternative nucleotide ispresent at that SNP position). Such high stringency conditions may bepreferable when using, for example, nucleic acid arrays ofallele-specific probes for SNP detection. Such high stringencyconditions are described in the preceding section, and are well known tothose skilled in the art and can be found in, for example, CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6.

In other embodiments, the arrays are used in conjunction withchemiluminescent detection technology. The following patents and patentapplications, which are all hereby incorporated by reference, provideadditional information pertaining to chemiluminescent detection: U.S.patent application Ser. Nos. 10/620,332 and 10/620,333 describechemiluminescent approaches for microarray detection; U.S. Pat. Nos.6,124,478, 6,107,024, 5,994,073, 5,981,768, 5,871,938, 5,843,681,5,800,999, and 5,773,628 describe methods and compositions of dioxetanefor performing chemiluminescent detection; and U.S. publishedapplication US2002/0110828 discloses methods and compositions formicroarray controls.

In one embodiment of the invention, a nucleic acid array can comprise anarray of probes of about 15-25 nucleotides in length. In furtherembodiments, a nucleic acid array can comprise any number of probes, inwhich at least one probe is capable of detecting one or more SNPsdisclosed in Table 1 and/or Table 2, and/or at least one probe comprisesa fragment of one of the sequences selected from the group consisting ofthose disclosed in Table 1, Table 2, the Sequence Listing, and sequencescomplementary thereto, said fragment comprising at least about 8consecutive nucleotides, preferably 10, 12, 15, 16, 18, 20, morepreferably 22, 25, 30, 40, 47, 50, 55, 60, 65, 70, 80, 90, 100, or moreconsecutive nucleotides (or any other number in-between) and containing(or being complementary to) a novel SNP allele disclosed in Table 1and/or Table 2. In some embodiments, the nucleotide complementary to theSNP site is within 5, 4, 3, 2, or 1 nucleotide from the center of theprobe, more preferably at the center of said probe.

A polynucleotide probe can be synthesized on the surface of thesubstrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application W095/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, V, mechanical or chemical bonding procedures. An array, such asthose described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more polynucleotides, or any other numberwhich lends itself to the efficient use of commercially availableinstrumentation.

Using such arrays or other kits/systems, the present invention providesmethods of identifying the SNPs disclosed herein in a test sample. Suchmethods typically involve incubating a test sample of nucleic acids withan array comprising one or more probes corresponding to at least one SNPposition of the present invention, and assaying for binding of a nucleicacid from the test sample with one or more of the probes. Conditions forincubating a SNP detection reagent (or a kit/system that employs one ormore such SNP detection reagents) with a test sample vary. Incubationconditions depend on such factors as the format employed in the assay,the detection methods employed, and the type and nature of the detectionreagents used in the assay. One skilled in the art will recognize thatany one of the commonly available hybridization, amplification and arrayassay formats can readily be adapted to detect the SNPs disclosedherein.

A SNP detection kit/system of the present invention may includecomponents that are used to prepare nucleic acids from a test sample forthe subsequent amplification and/or detection of a SNP-containingnucleic acid molecule. Such sample preparation components can be used toproduce nucleic acid extracts (including DNA and/or RNA), proteins ormembrane extracts from any bodily fluids (such as blood, serum, plasma,urine, saliva, phlegm, gastric juices, semen, tears, sweat, etc.), skin,hair, cells (especially nucleated cells), biopsies, buccal swabs ortissue specimens. The test samples used in the above-described methodswill vary based on such factors as the assay format, nature of thedetection method, and the specific tissues, cells or extracts used asthe test sample to be assayed. Methods of preparing nucleic acids,proteins, and cell extracts are well known in the art and can be readilyadapted to obtain a sample that is compatible with the system utilized.Automated sample preparation systems for extracting nucleic acids from atest sample are commercially available, and examples are Qiagen'sBioRobot 9600, Applied Biosystems' PRISM™ 6700 sample preparationsystem, and Roche Molecular Systems' COBAS AmpliPrep System.

Another form of kit contemplated by the present invention is acompartmentalized kit. A compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers include,for example, small glass containers, plastic containers, strips ofplastic, glass or paper, or arraying material such as silica. Suchcontainers allow one to efficiently transfer reagents from onecompartment to another compartment such that the test samples andreagents are not cross-contaminated, or from one container to anothervessel not included in the kit, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother or to another vessel. Such containers may include, for example,one or more containers which will accept the test sample, one or morecontainers which contain at least one probe or other SNP detectionreagent for detecting one or more SNPs of the present invention, one ormore containers which contain wash reagents (such as phosphate bufferedsaline, Tris-buffers, etc.), and one or more containers which containthe reagents used to reveal the presence of the bound probe or other SNPdetection reagents. The kit can optionally further comprise compartmentsand/or reagents for, for example, nucleic acid amplification or otherenzymatic reactions such as primer extension reactions, hybridization,ligation, electrophoresis (preferably capillary electrophoresis), massspectrometry, and/or laser-induced fluorescent detection. The kit mayalso include instructions for using the kit. Exemplary compartmentalizedkits include microfluidic devices known in the art (see, e.g., Weigl etal., “Lab-on-a-chip for drug development”, Adv Drug Deliv Rev. 2003 Feb.24; 55(3):349-77). In such microfluidic devices, the containers may bereferred to as, for example, microfluidic “compartments”, “chambers”, or“channels”.

Microfluidic devices, which may also be referred to as “lab-on-a-chip”systems, biomedical micro-electro-mechanical systems (bioMEMs), ormulticomponent integrated systems, are exemplary kits/systems of thepresent invention for analyzing SNPs. Such systems miniaturize andcompartmentalize processes such as probe/target hybridization, nucleicacid amplification, and capillary electrophoresis reactions in a singlefunctional device. Such microfluidic devices typically utilize detectionreagents in at least one aspect of the system, and such detectionreagents may be used to detect one or more SNPs of the presentinvention. One example of a microfluidic system is disclosed in U.S.Pat. No. 5,589,136, which describes the integration of PCR amplificationand capillary electrophoresis in chips. Exemplary microfluidic systemscomprise a pattern of microchannels designed onto a glass, silicon,quartz, or plastic wafer included on a microchip. The movements of thesamples may be controlled by electric, electroosmotic or hydrostaticforces applied across different areas of the microchip to createfunctional microscopic valves and pumps with no moving parts. Varyingthe voltage can be used as a means to control the liquid flow atintersections between the micro-machined channels and to change theliquid flow rate for pumping across different sections of the microchip.See, for example, U.S. Pat. No. 6,153,073, Dubrow et al., and U.S. Pat.No. 6,156,181, Parce et al.

For genotyping SNPs, an exemplary microfluidic system may integrate, forexample, nucleic acid amplification, primer extension, capillaryelectrophoresis, and a detection method such as laser inducedfluorescence detection. In a first step of an exemplary process forusing such an exemplary system, nucleic acid samples are amplified,preferably by PCR. Then, the amplification products are subjected toautomated primer extension reactions using ddNTPs (specific fluorescencefor each ddNTP) and the appropriate oligonucleotide primers to carry outprimer extension reactions which hybridize just upstream of the targetedSNP. Once the extension at the 3′ end is completed, the primers areseparated from the unincorporated fluorescent ddNTPs by capillaryelectrophoresis. The separation medium used in capillary electrophoresiscan be, for example, polyacrylamide, polyethyleneglycol or dextran. Theincorporated ddNTPs in the single nucleotide primer extension productsare identified by laser-induced fluorescence detection. Such anexemplary microchip can be used to process, for example, at least 96 to384 samples, or more, in parallel.

Uses of Nucleic Acid Molecules

The nucleic acid molecules of the present invention have a variety ofuses, especially in the diagnosis and treatment of CHD, and inparticular stenosis and MI. For example, the nucleic acid molecules areuseful as hybridization probes, such as for genotyping SNPs in messengerRNA, transcript, cDNA, genomic DNA, amplified DNA or other nucleic acidmolecules, and for isolating full-length cDNA and genomic clonesencoding the variant peptides disclosed in Table 1 as well as theirorthologs.

A probe can hybridize to any nucleotide sequence along the entire lengthof a nucleic acid molecule referred to in Table 1 and/or Table 2.Preferably, a probe of the present invention hybridizes to a region of atarget sequence that encompasses a SNP position indicated in Table 1and/or Table 2. More preferably, a probe hybridizes to a SNP-containingtarget sequence in a sequence-specific manner such that it distinguishesthe target sequence from other nucleotide sequences which vary from thetarget sequence only by which nucleotide is present at the SNP site.Such a probe is particularly useful for detecting the presence of aSNP-containing nucleic acid in a test sample, or for determining whichnucleotide (allele) is present at a particular SNP site (i.e.,genotyping the SNP site).

A nucleic acid hybridization probe may be used for determining thepresence, level, form, and/or distribution of nucleic acid expression.The nucleic acid whose level is determined can be DNA or RNA.Accordingly, probes specific for the SNPs described herein can be usedto assess the presence, expression and/or gene copy number in a givencell, tissue, or organism. These uses are relevant for diagnosis ofdisorders involving an increase or decrease in gene expression relativeto normal levels. In vitro techniques for detection of mRNA include, forexample, Northern blot hybridizations and in situ hybridizations. Invitro techniques for detecting DNA include Southern blot hybridizationsand in situ hybridizations (Sambrook and Russell, 2000, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y.).

Probes can be used as part of a diagnostic test kit for identifyingcells or tissues in which a variant protein is expressed, such as bymeasuring the level of a variant protein-encoding nucleic acid (e.g.,mRNA) in a sample of cells from a subject or determining if apolynucleotide contains a SNP of interest.

Thus, the nucleic acid molecules of the invention can be used ashybridization probes to detect the SNPs disclosed herein, therebydetermining whether an individual with the polymorphisms is at risk forstenosis or MI or has developed early stage stenosis or MI. Detection ofa SNP associated with a disease phenotype provides a diagnostic tool foran active disease and/or genetic predisposition to the disease.

Furthermore, the nucleic acid molecules of the invention are thereforeuseful for detecting a gene (gene information is disclosed in Table 2,for example) which contains a SNP disclosed herein and/or products ofsuch genes, such as expressed mRNA transcript molecules (transcriptinformation is disclosed in Table 1, for example), and are thus usefulfor detecting gene expression. The nucleic acid molecules can optionallybe implemented in, for example, an array or kit format for use indetecting gene expression.

The nucleic acid molecules of the invention are also useful as primersto amplify any given region of a nucleic acid molecule, particularly aregion containing a SNP identified in Table 1 and/or Table 2.

The nucleic acid molecules of the invention are also useful forconstructing recombinant vectors (described in greater detail below).Such vectors include expression vectors that express a portion of, orall of, any of the variant peptide sequences referred to in Table 1.Vectors also include insertion vectors, used to integrate into anothernucleic acid molecule sequence, such as into the cellular genome, toalter in situ expression of a gene and/or gene product. For example, anendogenous coding sequence can be replaced via homologous recombinationwith all or part of the coding region containing one or morespecifically introduced SNPs.

The nucleic acid molecules of the invention are also useful forexpressing antigenic portions of the variant proteins, particularlyantigenic portions that contain a variant amino acid sequence (e.g., anamino acid substitution) caused by a SNP disclosed in Table 1 and/orTable 2.

The nucleic acid molecules of the invention are also useful forconstructing vectors containing a gene regulatory region of the nucleicacid molecules of the present invention.

The nucleic acid molecules of the invention are also useful fordesigning ribozymes corresponding to all, or a part, of an mRNA moleculeexpressed from a SNP-containing nucleic acid molecule described herein.

The nucleic acid molecules of the invention are also useful forconstructing host cells expressing a part, or all, of the nucleic acidmolecules and variant peptides.

The nucleic acid molecules of the invention are also useful forconstructing transgenic animals expressing all, or a part, of thenucleic acid molecules and variant peptides. The production ofrecombinant cells and transgenic animals having nucleic acid moleculeswhich contain the SNPs disclosed in Table 1 and/or Table 2 allow, forexample, effective clinical design of treatment compounds and dosageregimens.

The nucleic acid molecules of the invention are also useful in assaysfor drug screening to identify compounds that, for example, modulatenucleic acid expression.

The nucleic acid molecules of the invention are also useful in genetherapy in patients whose cells have aberrant gene expression. Thus,recombinant cells, which include a patient's cells that have beenengineered ex vivo and returned to the patient, can be introduced intoan individual where the recombinant cells produce the desired protein totreat the individual.

SNP Genotyping Methods

The process of determining which specific nucleotide (i.e., allele) ispresent at each of one or more SNP positions, such as a SNP position ina nucleic acid molecule disclosed in Table 1 and/or Table 2, is referredto as SNP genotyping. The present invention provides methods of SNPgenotyping, such as for use in screening for CHD, and in particularstenosis and MI, or related pathologies, or determining predispositionthereto, or determining responsiveness to a form of treatment, or ingenome mapping or SNP association analysis, etc.

Nucleic acid samples can be genotyped to determine which allele(s)is/are present at any given genetic region (e.g., SNP position) ofinterest by methods well known in the art. The neighboring sequence canbe used to design SNP detection reagents such as oligonucleotide probes,which may optionally be implemented in a kit format. Exemplary SNPgenotyping methods are described in Chen et al., “Single nucleotidepolymorphism genotyping: biochemistry, protocol, cost and throughput”,Pharmacogenomics J. 2003; 3(2):77-96; Kwok et al., “Detection of singlenucleotide polymorphisms”, Curr Issues Mol Biol. 2003 April; 5(2):43-60;Shi, “Technologies for individual genotyping: detection of geneticpolymorphisms in drug targets and disease genes”, Am J Pharmacogenomics.2002; 2(3):197-205; and Kwok, “Methods for genotyping single nucleotidepolymorphisms”, Annu Rev Genomics Hum Genet 2001; 2:235-58. Exemplarytechniques for high-throughput SNP genotyping are described inMarnellos, “High-throughput SNP analysis for genetic associationstudies”, Curr Opin Drug Discov Devel. 2003 May; 6(3):317-21. Common SNPgenotyping methods include, but are not limited to, TaqMan assays,molecular beacon assays, nucleic acid arrays, allele-specific primerextension, allele-specific PCR, arrayed primer extension, homogeneousprimer extension assays, primer extension with detection by massspectrometry, pyrosequencing, multiplex primer extension sorted ongenetic arrays, ligation with rolling circle amplification, homogeneousligation, OLA (U.S. Pat. No. 4,988,167), multiplex ligation reactionsorted on genetic arrays, restriction-fragment length polymorphism,single base extension-tag assays, and the Invader assay. Such methodsmay be used in combination with detection mechanisms such as, forexample, luminescence or chemiluminescence detection, fluorescencedetection, time-resolved fluorescence detection, fluorescence resonanceenergy transfer, fluorescence polarization, mass spectrometry, andelectrical detection.

Various methods for detecting polymorphisms include, but are not limitedto, methods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science230:1242 (1985); Cotton et al., PNAS 85:4397 (1988); and Saleeba et al.,Meth. Enzymol. 217:286-295 (1992)), comparison of the electrophoreticmobility of variant and wild type nucleic acid molecules (Orita et al.,PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); andHayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and assayingthe movement of polymorphic or wild-type fragments in polyacrylamidegels containing a gradient of denaturant using denaturing gradient gelelectrophoresis (DGGE) (Myers et al., Nature 313:495 (1985)). Sequencevariations at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or chemical cleavagemethods.

In a preferred embodiment, SNP genotyping is performed using the TaqManassay, which is also known as the 5′ nuclease assay (U.S. Pat. Nos.5,210,015 and 5,538,848). The TaqMan assay detects the accumulation of aspecific amplified product during PCR. The TaqMan assay utilizes anoligonucleotide probe labeled with a fluorescent reporter dye and aquencher dye. The reporter dye is excited by irradiation at anappropriate wavelength, it transfers energy to the quencher dye in thesame probe via a process called fluorescence resonance energy transfer(FRET). When attached to the probe, the excited reporter dye does notemit a signal. The proximity of the quencher dye to the reporter dye inthe intact probe maintains a reduced fluorescence for the reporter. Thereporter dye and quencher dye may be at the 5′ most and the 3′ mostends, respectively, or vice versa. Alternatively, the reporter dye maybe at the 5′ or 3′ most end while the quencher dye is attached to aninternal nucleotide, or vice versa. In yet another embodiment, both thereporter and the quencher may be attached to internal nucleotides at adistance from each other such that fluorescence of the reporter isreduced.

During PCR, the 5′ nuclease activity of DNA polymerase cleaves theprobe, thereby separating the reporter dye and the quencher dye andresulting in increased fluorescence of the reporter. Accumulation of PCRproduct is detected directly by monitoring the increase in fluorescenceof the reporter dye. The DNA polymerase cleaves the probe between thereporter dye and the quencher dye only if the probe hybridizes to thetarget SNP-containing template which is amplified during PCR, and theprobe is designed to hybridize to the target SNP site only if aparticular SNP allele is present.

Preferred TaqMan primer and probe sequences can readily be determinedusing the SNP and associated nucleic acid sequence information providedherein. A number of computer programs, such as Primer Express (AppliedBiosystems, Foster City, Calif.), can be used to rapidly obtain optimalprimer/probe sets. It will be apparent to one of skill in the art thatsuch primers and probes for detecting the SNPs of the present inventionare useful in diagnostic assays for CHD, and in particular stenosis andMI and related pathologies, and can be readily incorporated into a kitformat. The present invention also includes modifications of the Taqmanassay well known in the art such as the use of Molecular Beacon probes(U.S. Pat. Nos. 5,118,801 and 5,312,728) and other variant formats (U.S.Pat. Nos. 5,866,336 and 6,117,635).

Another preferred method for genotyping the SNPs of the presentinvention is the use of two oligonucleotide probes in an OLA (see, e.g.,U.S. Pat. No. 4,988,617). In this method, one probe hybridizes to asegment of a target nucleic acid with its 3′ most end aligned with theSNP site. A second probe hybridizes to an adjacent segment of the targetnucleic acid molecule directly 3′ to the first probe. The two juxtaposedprobes hybridize to the target nucleic acid molecule, and are ligated inthe presence of a linking agent such as a ligase if there is perfectcomplementarity between the 3′ most nucleotide of the first probe withthe SNP site. If there is a mismatch, ligation would not occur. Afterthe reaction, the ligated probes are separated from the target nucleicacid molecule, and detected as indicators of the presence of a SNP.

The following patents, patent applications, and published internationalpatent applications, which are all hereby incorporated by reference,provide additional information pertaining to techniques for carrying outvarious types of OLA: U.S. Pat. Nos. 6,027,889, 6,268,148, 5,494,810,5,830,711, and 6,054,564 describe OLA strategies for performing SNPdetection; WO 97/31256 and WO 00/56927 describe OLA strategies forperforming SNP detection using universal arrays, wherein a zipcodesequence can be introduced into one of the hybridization probes, and theresulting product, or amplified product, hybridized to a universal zipcode array; U.S. application Ser. No. 01/17329 (and Ser. No. 09/584,905)describes OLA (or LDR) followed by PCR, wherein zipcodes areincorporated into OLA probes, and amplified PCR products are determinedby electrophoretic or universal zipcode array readout; U.S. applications60/427,818, 60/445,636, and 60/445,494 describe SNPlex methods andsoftware for multiplexed SNP detection using OLA followed by PCR,wherein zipcodes are incorporated into OLA probes, and amplified PCRproducts are hybridized with a zipchute reagent, and the identity of theSNP determined from electrophoretic readout of the zipchute. In someembodiments, OLA is carried out prior to PCR (or another method ofnucleic acid amplification). In other embodiments, PCR (or anothermethod of nucleic acid amplification) is carried out prior to OLA.

Another method for SNP genotyping is based on mass spectrometry. Massspectrometry takes advantage of the unique mass of each of the fournucleotides of DNA. SNPs can be unambiguously genotyped by massspectrometry by measuring the differences in the mass of nucleic acidshaving alternative SNP alleles. MALDI-TOF (Matrix Assisted LaserDesorption Ionization—Time of Flight) mass spectrometry technology ispreferred for extremely precise determinations of molecular mass, suchas SNPs. Numerous approaches to SNP analysis have been developed basedon mass spectrometry. Preferred mass spectrometry-based methods of SNPgenotyping include primer extension assays, which can also be utilizedin combination with other approaches, such as traditional gel-basedformats and microarrays.

Typically, the primer extension assay involves designing and annealing aprimer to a template PCR amplicon upstream (5′) from a target SNPposition. A mix of dideoxynucleotide triphosphates (ddNTPs) and/ordeoxynucleotide triphosphates (dNTPs) are added to a reaction mixturecontaining template (e.g., a SNP-containing nucleic acid molecule whichhas typically been amplified, such as by PCR), primer, and DNApolymerase. Extension of the primer terminates at the first position inthe template where a nucleotide complementary to one of the ddNTPs inthe mix occurs. The primer can be either immediately adjacent (i.e., thenucleotide at the 3′ end of the primer hybridizes to the nucleotide nextto the target SNP site) or two or more nucleotides removed from the SNPposition. If the primer is several nucleotides removed from the targetSNP position, the only limitation is that the template sequence betweenthe 3′ end of the primer and the SNP position cannot contain anucleotide of the same type as the one to be detected, or this willcause premature termination of the extension primer. Alternatively, ifall four ddNTPs alone, with no dNTPs, are added to the reaction mixture,the primer will always be extended by only one nucleotide, correspondingto the target SNP position. In this instance, primers are designed tobind one nucleotide upstream from the SNP position (i.e., the nucleotideat the 3′ end of the primer hybridizes to the nucleotide that isimmediately adjacent to the target SNP site on the 5′ side of the targetSNP site). Extension by only one nucleotide is preferable, as itminimizes the overall mass of the extended primer, thereby increasingthe resolution of mass differences between alternative SNP nucleotides.Furthermore, mass-tagged ddNTPs can be employed in the primer extensionreactions in place of unmodified ddNTPs. This increases the massdifference between primers extended with these ddNTPs, thereby providingincreased sensitivity and accuracy, and is particularly useful fortyping heterozygous base positions. Mass-tagging also alleviates theneed for intensive sample-preparation procedures and decreases thenecessary resolving power of the mass spectrometer.

The extended primers can then be purified and analyzed by MALDI-TOF massspectrometry to determine the identity of the nucleotide present at thetarget SNP position. In one method of analysis, the products from theprimer extension reaction are combined with light absorbing crystalsthat form a matrix. The matrix is then hit with an energy source such asa laser to ionize and desorb the nucleic acid molecules into thegas-phase. The ionized molecules are then ejected into a flight tube andaccelerated down the tube towards a detector. The time between theionization event, such as a laser pulse, and collision of the moleculewith the detector is the time of flight of that molecule. The time offlight is precisely correlated with the mass-to-charge ratio (m/z) ofthe ionized molecule. Ions with smaller m/z travel down the tube fasterthan ions with larger m/z and therefore the lighter ions reach thedetector before the heavier ions. The time-of-flight is then convertedinto a corresponding, and highly precise, m/z. In this manner, SNPs canbe identified based on the slight differences in mass, and thecorresponding time of flight differences, inherent in nucleic acidmolecules having different nucleotides at a single base position. Forfurther information regarding the use of primer extension assays inconjunction with MALDI-TOF mass spectrometry for SNP genotyping, see,e.g., Wise et al., “A standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry”, Rapid CommunMass Spectrom. 2003; 17(11): 1195-202.

The following references provide further information describing massspectrometry-based methods for SNP genotyping: Bocker, “SNP and mutationdiscovery using base-specific cleavage and MALDI-TOF mass spectrometry”,Bioinformatics. 2003 July; 19 Suppl 1:144-153; Storm et al., “MALDI-TOFmass spectrometry-based SNP genotyping”, Methods Mol. Biol. 2003;212:241-62; Jurinke et al., “The use of Mass ARRAY technology for highthroughput genotyping”, Adv Biochem Eng Biotechnol. 2002; 77:57-74; andJurinke et al., “Automated genotyping using the DNA MassArraytechnology”, Methods Mol. Biol. 2002; 187:179-92.

SNPs can also be scored by direct DNA sequencing. A variety of automatedsequencing procedures can be utilized ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO94/16101; Cohen et al., Adv. Chromatogr. 36:127-162(1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159(1993)). The nucleic acid sequences of the present invention enable oneof ordinary skill in the art to readily design sequencing primers forsuch automated sequencing procedures. Commercial instrumentation, suchas the Applied Biosystems 377, 3100, 3700, 3730, and 3730x1 DNAAnalyzers (Foster City, Calif.), is commonly used in the art forautomated sequencing.

Other methods that can be used to genotype the SNPs of the presentinvention include single-strand conformational polymorphism (SSCP), anddenaturing gradient gel electrophoresis (DGGE) (Myers et al., Nature313:495 (1985)). SSCP identifies base differences by alteration inelectrophoretic migration of single stranded PCR products, as describedin Orita et al., Proc. Nat. Acad. Single-stranded PCR products can begenerated by heating or otherwise denaturing double stranded PCRproducts. Single-stranded nucleic acids may refold or form secondarystructures that are partially dependent on the base sequence. Thedifferent electrophoretic mobilities of single-stranded amplificationproducts are related to base-sequence differences at SNP positions. DGGEdifferentiates SNP alleles based on the different sequence-dependentstabilities and melting properties inherent in polymorphic DNA and thecorresponding differences in electrophoretic migration patterns in adenaturing gradient gel (Erlich, ed., PCR Technology, Principles andApplications for DNA Amplification, W.H. Freeman and Co, New York, 1992,Chapter 7).

Sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can also be usedto score SNPs based on the development or loss of a ribozyme cleavagesite. Perfectly matched sequences can be distinguished from mismatchedsequences by nuclease cleavage digestion assays or by differences inmelting temperature. If the SNP affects a restriction enzyme cleavagesite, the SNP can be identified by alterations in restriction enzymedigestion patterns, and the corresponding changes in nucleic acidfragment lengths determined by gel electrophoresis

SNP genotyping can include the steps of, for example, collecting abiological sample from a human subject (e.g., sample of tissues, cells,fluids, secretions, etc.), isolating nucleic acids (e.g., genomic DNA,mRNA or both) from the cells of the sample, contacting the nucleic acidswith one or more primers which specifically hybridize to a region of theisolated nucleic acid containing a target SNP under conditions such thathybridization and amplification of the target nucleic acid regionoccurs, and determining the nucleotide present at the SNP position ofinterest, or, in some assays, detecting the presence or absence of anamplification product (assays can be designed so that hybridizationand/or amplification will only occur if a particular SNP allele ispresent or absent). In some assays, the size of the amplificationproduct is detected and compared to the length of a control sample; forexample, deletions and insertions can be detected by a change in size ofthe amplified product compared to a normal genotype.

SNP genotyping is useful for numerous practical applications, asdescribed below. Examples of such applications include, but are notlimited to, SNP-disease association analysis, disease predispositionscreening, disease diagnosis, disease prognosis, disease progressionmonitoring, determining therapeutic strategies based on an individual'sgenotype (“pharmacogenomics”), developing therapeutic agents based onSNP genotypes associated with a disease or likelihood of responding to adrug, stratifying a patient population for clinical trial for atreatment regimen, predicting the likelihood that an individual willexperience toxic side effects from a therapeutic agent, and humanidentification applications such as forensics.

Analysis of Genetic Association Between SNPs and Phenotypic Traits

SNP genotyping for disease diagnosis, disease predisposition screening,disease prognosis, determining drug responsiveness (pharmacogenomics),drug toxicity screening, and other uses described herein, typicallyrelies on initially establishing a genetic association between one ormore specific SNPs and the particular phenotypic traits of interest.

Different study designs may be used for genetic association studies(Modern Epidemiology, Lippincott Williams & Wilkins (1998), 609-622).Observational studies are most frequently carried out in which theresponse of the patients is not interfered with. The first type ofobservational study identifies a sample of persons in whom the suspectedcause of the disease is present and another sample of persons in whomthe suspected cause is absent, and then the frequency of development ofdisease in the two samples is compared. These sampled populations arecalled cohorts, and the study is a prospective study. The other type ofobservational study is case-control or a retrospective study. In typicalcase-control studies, samples are collected from individuals with thephenotype of interest (cases) such as certain manifestations of adisease, and from individuals without the phenotype (controls) in apopulation (target population) that conclusions are to be drawn from.Then the possible causes of the disease are investigatedretrospectively. As the time and costs of collecting samples incase-control studies are considerably less than those for prospectivestudies, case-control studies are the more commonly used study design ingenetic association studies, at least during the exploration anddiscovery stage.

In both types of observational studies, there may be potentialconfounding factors that should be taken into consideration. Confoundingfactors are those that are associated with both the real cause(s) of thedisease and the disease itself, and they include demographic informationsuch as age, gender, ethnicity as well as environmental factors. Whenconfounding factors are not matched in cases and controls in a study,and are not controlled properly, spurious association results can arise.If potential confounding factors are identified, they should becontrolled for by analysis methods explained below.

In a genetic association study, the cause of interest to be tested is acertain allele or a SNP or a combination of alleles or a haplotype fromseveral SNPs. Thus, tissue specimens (e.g., whole blood) from thesampled individuals may be collected and genomic DNA genotyped for theSNP(s) of interest. In addition to the phenotypic trait of interest,other information such as demographic (e.g., age, gender, ethnicity,etc.), clinical, and environmental information that may influence theoutcome of the trait can be collected to further characterize and definethe sample set. In many cases, these factors are known to be associatedwith diseases and/or SNP allele frequencies. There are likelygene-environment and/or gene-gene interactions as well. Analysis methodsto address gene-environment and gene-gene interactions (for example, theeffects of the presence of both susceptibility alleles at two differentgenes can be greater than the effects of the individual alleles at twogenes combined) are discussed below.

After all the relevant phenotypic and genotypic information has beenobtained, statistical analyses are carried out to determine if there isany significant correlation between the presence of an allele or agenotype with the phenotypic characteristics of an individual.Preferably, data inspection and cleaning are first performed beforecarrying out statistical tests for genetic association. Epidemiologicaland clinical data of the samples can be summarized by descriptivestatistics with tables and graphs. Data validation is preferablyperformed to check for data completion, inconsistent entries, andoutliers. Chi-squared tests and t-tests (Wilcoxon rank-sum tests ifdistributions are not normal) may then be used to check for significantdifferences between cases and controls for discrete and continuousvariables, respectively. To ensure genotyping quality, Hardy-Weinbergdisequilibrium tests can be performed on cases and controls separately.Significant deviation from Hardy-Weinberg equilibrium (HWE) in bothcases and controls for individual markers can be indicative ofgenotyping errors. If HWE is violated in a majority of markers, it isindicative of population substructure that should be furtherinvestigated. Moreover, Hardy-Weinberg disequilibrium in cases only canindicate genetic association of the markers with the disease (GeneticData Analysis, Weir B., Sinauer (1990)).

To test whether an allele of a single SNP is associated with the case orcontrol status of a phenotypic trait, one skilled in the art can compareallele frequencies in cases and controls. Standard chi-squared tests andFisher exact tests can be carried out on a 2×2 table (2 SNP alleles×2outcomes in the categorical trait of interest). To test whethergenotypes of a SNP are associated, chi-squared tests can be carried outon a 3×2 table (3 genotypes×2 outcomes). Score tests are also carriedout for genotypic association to contrast the three genotypicfrequencies (major homozygotes, heterozygotes and minor homozygotes) incases and controls, and to look for trends using 3 different modes ofinheritance, namely dominant (with contrast coefficients 2, −1, −1),additive (with contrast coefficients 1, 0, −1) and recessive (withcontrast coefficients 1, 1, −2). Odds ratios for minor versus majoralleles, and odds ratios for heterozygote and homozygote variants versusthe wild type genotypes are calculated with the desired confidencelimits, usually 95%.

In order to control for confounders and to test for interaction andeffect modifiers, stratified analyses may be performed using stratifiedfactors that are likely to be confounding, including demographicinformation such as age, ethnicity, and gender, or an interactingelement or effect modifier, such as a known major gene (e.g., APOE forAlzheimer's disease or HLA genes for autoimmune diseases), orenvironmental factors such as smoking in lung cancer. Stratifiedassociation tests may be carried out using Cochran-Mantel-Haenszel teststhat take into account the ordinal nature of genotypes with 0, 1, and 2variant alleles. Exact tests by StatXact may also be performed whencomputationally possible. Another way to adjust for confounding effectsand test for interactions is to perform stepwise multiple logisticregression analysis using statistical packages such as SAS or R.Logistic regression is a model-building technique in which the bestfitting and most parsimonious model is built to describe the relationbetween the dichotomous outcome (for instance, getting a certain diseaseor not) and a set of independent variables (for instance, genotypes ofdifferent associated genes, and the associated demographic andenvironmental factors). The most common model is one in which the logittransformation of the odds ratios is expressed as a linear combinationof the variables (main effects) and their cross-product terms(interactions) (Applied Logistic Regression, Hosmer and Lemeshow, Wiley(2000)). To test whether a certain variable or interaction issignificantly associated with the outcome, coefficients in the model arefirst estimated and then tested for statistical significance of theirdeparture from zero.

In addition to performing association tests one marker at a time,haplotype association analysis may also be performed to study a numberof markers that are closely linked together. Haplotype association testscan have better power than genotypic or allelic association tests whenthe tested markers are not the disease-causing mutations themselves butare in linkage disequilibrium with such mutations. The test will even bemore powerful if the disease is indeed caused by a combination ofalleles on a haplotype (e.g., APOE is a haplotype formed by 2 SNPs thatare very close to each other). In order to perform haplotype associationeffectively, marker-marker linkage disequilibrium measures, both D′ andr², are typically calculated for the markers within a gene to elucidatethe haplotype structure. Recent studies (Daly et al, Nature Genetics,29, 232-235, 2001) in linkage disequilibrium indicate that SNPs within agene are organized in block pattern, and a high degree of linkagedisequilibrium exists within blocks and very little linkagedisequilibrium exists between blocks. Haplotype association with thedisease status can be performed using such blocks once they have beenelucidated.

Haplotype association tests can be carried out in a similar fashion asthe allelic and genotypic association tests. Each haplotype in a gene isanalogous to an allele in a multi-allelic marker. One skilled in the artcan either compare the haplotype frequencies in cases and controls ortest genetic association with different pairs of haplotypes. It has beenproposed (Schaid et al, Am. J. Hum. Genet., 70, 425-434, 2002) thatscore tests can be done on haplotypes using the program “haplo.score.”In that method, haplotypes are first inferred by EM algorithm and scoretests are carried out with a generalized linear model (GLM) frameworkthat allows the adjustment of other factors.

An important decision in the performance of genetic association tests isthe determination of the significance level at which significantassociation can be declared when the P value of the tests reaches thatlevel. In an exploratory analysis where positive hits will be followedup in subsequent confirmatory testing, an unadjusted P value<0.2 (asignificance level on the lenient side), for example, may be used forgenerating hypotheses for significant association of a SNP with certainphenotypic characteristics of a disease. It is preferred that ap-value<0.05 (a significance level traditionally used in the art) isachieved in order for a SNP to be considered to have an association witha disease. It is more preferred that a p-value<0.01 (a significancelevel on the stringent side) is achieved for an association to bedeclared. When hits are followed up in confirmatory analyses in moresamples of the same source or in different samples from differentsources, adjustment for multiple testing will be performed as to avoidexcess number of hits while maintaining the experiment-wide error ratesat 0.05. While there are different methods to adjust for multipletesting to control for different kinds of error rates, a commonly usedbut rather conservative method is Bonferroni correction to control theexperiment-wise or family-wise error rate (Multiple comparisons andmultiple tests, Westfall et al, SAS Institute (1999)). Permutation teststo control for the false discovery rates, FDR, can be more powerful(Benjamini and Hochberg, Journal of the Royal Statistical Society,Series B 57, 1289-1300, 1995, Resampling-based Multiple Testing,Westfall and Young, Wiley (1993)). Such methods to control formultiplicity would be preferred when the tests are dependent andcontrolling for false discovery rates is sufficient as opposed tocontrolling for the experiment-wise error rates.

In replication studies using samples from different populations afterstatistically significant markers have been identified in theexploratory stage, meta-analyses can then be performed by combiningevidence of different studies (Modern Epidemiology, Lippincott Williams& Wilkins, 1998, 643-673). If available, association results known inthe art for the same SNPs can be included in the meta-analyses.

Since both genotyping and disease status classification can involveerrors, sensitivity analyses may be performed to see how odds ratios andp-values would change upon various estimates on genotyping and diseaseclassification error rates.

It has been well known that subpopulation-based sampling bias betweencases and controls can lead to spurious results in case-controlassociation studies (Ewens and Spielman, Am. J. Hum. Genet. 62, 450-458,1995) when prevalence of the disease is associated with differentsubpopulation groups. Such bias can also lead to a loss of statisticalpower in genetic association studies. To detect populationstratification, Pritchard and Rosenberg (Pritchard et al. Am. J. Hum.Gen. 1999, 65:220-228) suggested typing markers that are unlinked to thedisease and using results of association tests on those markers todetermine whether there is any population stratification. Whenstratification is detected, the genomic control (GC) method as proposedby Devlin and Roeder (Devlin et al. Biometrics 1999, 55:997-1004) can beused to adjust for the inflation of test statistics due to populationstratification. GC method is robust to changes in population structurelevels as well as being applicable to DNA pooling designs (Devlin et al.Genet. Epidem. 20001, 21:273-284).

While Pritchard's method recommended using 15-20 unlinked microsatellitemarkers, it suggested using more than 30 biallelic markers to get enoughpower to detect population stratification. For the GC method, it hasbeen shown (Bacanu et al. Am. J. Hum. Genet. 2000, 66:1933-1944) thatabout 60-70 biallelic markers are sufficient to estimate the inflationfactor for the test statistics due to population stratification. Hence,70 intergenic SNPs can be chosen in unlinked regions as indicated in agenome scan (Kehoe et al. Hum. Mol. Genet. 1999, 8:237-245).

Once individual risk factors, genetic or non-genetic, have been foundfor the predisposition to disease, the next step is to set up aclassification/prediction scheme to predict the category (for instance,disease or no-disease) that an individual will be in depending on hisgenotypes of associated SNPs and other non-genetic risk factors.Logistic regression for discrete trait and linear regression forcontinuous trait are standard techniques for such tasks (AppliedRegression Analysis, Draper and Smith, Wiley (1998)). Moreover, othertechniques can also be used for setting up classification. Suchtechniques include, but are not limited to, MART, CART, neural network,and discriminant analyses that are suitable for use in comparing theperformance of different methods (The Elements of Statistical Learning,Hastie, Tibshirani & Friedman, Springer (2002)).

Disease Diagnosis and Predisposition Screening

Information on association/correlation between genotypes anddisease-related phenotypes can be exploited in several ways. Forexample, in the case of a highly statistically significant associationbetween one or more SNPs with predisposition to a disease for whichtreatment is available, detection of such a genotype pattern in anindividual may justify immediate administration of treatment, or atleast the institution of regular monitoring of the individual. Detectionof the susceptibility alleles associated with serious disease in acouple contemplating having children may also be valuable to the couplein their reproductive decisions. In the case of a weaker but stillstatistically significant association between a SNP and a human disease,immediate therapeutic intervention or monitoring may not be justifiedafter detecting the susceptibility allele or SNP. Nevertheless, thesubject can be motivated to begin simple life-style changes (e.g., diet,exercise) that can be accomplished at little or no cost to theindividual but would confer potential benefits in reducing the risk ofdeveloping conditions for which that individual may have an increasedrisk by virtue of having the risk allele(s).

The SNPs of the invention may contribute to the development of CHD in anindividual in different ways. Some polymorphisms occur within a proteincoding sequence and contribute to disease phenotype by affecting proteinstructure. Other polymorphisms occur in noncoding regions but may exertphenotypic effects indirectly via influence on, for example,replication, transcription, and/or translation. A single SNP may affectmore than one phenotypic trait. Likewise, a single phenotypic trait maybe affected by multiple SNPs in different genes.

As used herein, the terms “diagnose,” “diagnosis,” and “diagnostics”include, but are not limited to any of the following: detection of CHDsuch as stenosis and MI that an individual may presently have,predisposition/susceptibility screening (i.e., determining the increasedrisk of an individual in developing stenosis or MI in the future, ordetermining whether an individual has a decreased risk of developing CHDin the future), determining a particular type or subclass of CHD in anindividual known to have CHD, confirming or reinforcing a previouslymade diagnosis of CHD, pharmacogenomic evaluation of an individual todetermine which therapeutic strategy that individual is most likely topositively respond to or to predict whether a patient is likely torespond to a particular treatment such as statins, predicting whether apatient is likely to experience toxic effects from a particulartreatment or therapeutic compound, and evaluating the future prognosisof an individual having CHD. Such diagnostic uses are based on the SNPsindividually or in a unique combination or SNP haplotypes of the presentinvention.

Haplotypes are particularly useful in that, for example, fewer SNPs canbe genotyped to determine if a particular genomic region harbors a locusthat influences a particular phenotype, such as in linkagedisequilibrium-based SNP association analysis.

Linkage disequilibrium (LD) refers to the co-inheritance of alleles(e.g., alternative nucleotides) at two or more different SNP sites atfrequencies greater than would be expected from the separate frequenciesof occurrence of each allele in a given population. The expectedfrequency of co-occurrence of two alleles that are inheritedindependently is the frequency of the first allele multiplied by thefrequency of the second allele. Alleles that co-occur at expectedfrequencies are said to be in “linkage equilibrium.” In contrast, LDrefers to any non-random genetic association between allele(s) at two ormore different SNP sites, which is generally due to the physicalproximity of the two loci along a chromosome. LD can occur when two ormore SNPs sites are in close physical proximity to each other on a givenchromosome and therefore alleles at these SNP sites will tend to remainunseparated for multiple generations with the consequence that aparticular nucleotide (allele) at one SNP site will show a non-randomassociation with a particular nucleotide (allele) at a different SNPsite located nearby. Hence, genotyping one of the SNP sites will givealmost the same information as genotyping the other SNP site that is inLD.

Various degrees of LD can be encountered between two or more SNPs withthe result being that some SNPs are more closely associated (i.e., instronger LD) than others. Furthermore, the physical distance over whichLD extends along a chromosome differs between different regions of thegenome, and therefore the degree of physical separation between two ormore SNP sites necessary for LD to occur can differ between differentregions of the genome.

For diagnostic purposes and similar uses, if a particular SNP site isfound to be useful for diagnosing CHD (e.g., has a significantstatistical association with the condition and/or is recognized as acausative polymorphism for the condition), then the skilled artisanwould recognize that other SNP sites which are in LD with this SNP sitewould also be useful for diagnosing the condition. Thus, polymorphisms(e.g., SNPs and/or haplotypes) that are not the actual disease-causing(causative) polymorphisms, but are in LD with such causativepolymorphisms, are also useful. In such instances, the genotype of thepolymorphism(s) that is/are in LD with the causative polymorphism ispredictive of the genotype of the causative polymorphism and,consequently, predictive of the phenotype (e.g., CHD) that is influencedby the causative SNP(s). Therefore, polymorphic markers that are in LDwith causative polymorphisms are useful as diagnostic markers, and areparticularly useful when the actual causative polymorphism(s) is/areunknown.

Examples of polymorphisms that can be in LD with one or more causativepolymorphisms (and/or in LD with one or more polymorphisms that have asignificant statistical association with a condition) and thereforeuseful for diagnosing the same condition that the causative/associatedSNP(s) is used to diagnose, include other SNPs in the same gene,protein-coding, or mRNA transcript-coding region as thecausative/associated SNP, other SNPs in the same exon or same intron asthe causative/associated SNP, other SNPs in the same haplotype block asthe causative/associated SNP, other SNPs in the same intergenic regionas the causative/associated SNP, SNPs that are outside but near a gene(e.g., within 6 kb on either side, 5′ or 3′, of a gene boundary) thatharbors a causative/associated SNP, etc. Such useful LD SNPs can beselected from among the SNPs disclosed in Tables 1-2, for example.

Linkage disequilibrium in the human genome is reviewed in: Wall et al.,“Haplotype blocks and linkage disequilibrium in the human genome”, NatRev Genet. 2003 August; 4(8):587-97; Garner et al., “On selectingmarkers for association studies: patterns of linkage disequilibriumbetween two and three diallelic loci”, Genet Epidemiol. 2003 January;24(1):57-67; Ardlie et al., “Patterns of linkage disequilibrium in thehuman genome”, Nat Rev Genet. 2002 April; 3(4):299-309 (erratum in NatRev Genet 2002 July; 3(7):566); and Remm et al., “High-densitygenotyping and linkage disequilibrium in the human genome usingchromosome 22 as a model”; Curr Opin Chem Biol. 2002 February;6(1):24-30; Haldane J B S (1919) The combination of linkage values, andthe calculation of distances between the loci of linked factors. J Genet8:299-309; Mendel, G. (1866) Versuche über Pflanzen-Hybriden.Verhandlungen des naturforschenden Vereines in Brünn [Proceedings of theNatural History Society of Brünn]; Lewin B (1990) Genes IV. OxfordUniversity Press, New York, USA; Hartl D L and Clark A G (1989)Principles of Population Genetics 2^(nd) ed. Sinauer Associates, Inc.Sunderland, Mass., USA; Gillespie J H (2004) Population Genetics: AConcise Guide. 2^(nd) ed. Johns Hopkins University Press. USA; LewontinR C (1964) The interaction of selection and linkage. I. Generalconsiderations; heterotic models. Genetics 49:49-67; Hoel P G (1954)Introduction to Mathematical Statistics 2^(nd) ed. John Wiley & Sons,Inc. New York, USA; Hudson R R (2001) Two-locus sampling distributionsand their application. Genetics 159:1805-1817; Dempster A P, Laird N M,Rubin D B (1977) Maximum likelihood from incomplete data via the EMalgorithm. J R Stat Soc 39:1-38; Excoffier L, Slatkin M (1995)Maximum-likelihood estimation of molecular haplotype frequencies in adiploid population. Mol Biol Evol 12(5):921-927; Tregouet D A, EscolanoS, Tiret L, Mallet A, Golmard J L (2004) A new algorithm forhaplotype-based association analysis: the Stochastic-EM algorithm. AnnHum Genet 68(Pt 2):165-177; Long A D and Langley C H (1999) The power ofassociation studies to detect the contribution of candidate genetic locito variation in complex traits. Genome Research 9:720-731; Agresti A(1990) Categorical Data Analysis. John Wiley & Sons, Inc. New York, USA;Lange K (1997) Mathematical and Statistical Methods for GeneticAnalysis. Springer-Verlag New York, Inc. New York, USA; TheInternational HapMap Consortium (2003) The International HapMap Project.Nature 426:789-796; The International HapMap Consortium (2005) Ahaplotype map of the human genome. Nature 437:1299-1320; Thorisson G A,Smith A V, Krishnan L, Stein L D (2005), The International HapMapProject Web Site. Genome Research 15:1591-1593; McVean G, Spencer C C A,Chaix R (2005) Perspectives on human genetic variation from the HapMapproject. PLoS Genetics 1(4):413-418; Hirschhorn J N, Daly M J (2005)Genome-wide association studies for common diseases and complex traits.Nat Genet 6:95-108; Schrodi S J (2005) A probabilistic approach tolarge-scale association scans: a semi-Bayesian method to detectdisease-predisposing alleles. SAGMB 4(1):31; Wang W Y S, Barratt B J,Clayton D G, Todd J A (2005) Genome-wide association studies:theoretical and practical concerns. Nat Rev Genet 6:109-118. Pritchard JK, Przeworski M (2001) Linkage disequilibrium in humans: models anddata. Am J Hum Genet 69:1-14.

As discussed above, one aspect of the present invention is the discoverythat SNPs which are in certain LD distance with the interrogated SNP canalso be used as valid markers for identifying an increased or decreasedrisks of having or developing CHD. As used herein, the term“interrogated SNP” refers to SNPs that have been found to be associatedwith an increased or decreased risk of disease using genotyping resultsand analysis, or other appropriate experimental method as exemplified inthe working examples described in this application. As used herein, theterm “LD SNP” refers to a SNP that has been characterized as a SNPassociating with an increased or decreased risk of diseases due to theirbeing in LD with the “interrogated SNP” under the methods of calculationdescribed in the application. Below, applicants describe the methods ofcalculation with which one of ordinary skilled in the art may determineif a particular SNP is in LD with an interrogated SNP. The parameter r²is commonly used in the genetics art to characterize the extent oflinkage disequilibrium between markers (Hudson, 2001). As used herein,the term “in LD with” refers to a particular SNP that is measured atabove the threshold of a parameter such as r² with an interrogated SNP.

It is now common place to directly observe genetic variants in a sampleof chromosomes obtained from a population. Suppose one has genotype dataat two genetic markers located on the same chromosome, for the markers Aand B. Further suppose that two alleles segregate at each of these twomarkers such that alleles A₁ and A₂ can be found at marker A and allelesB₁ and B₂ at marker B. Also assume that these two markers are on a humanautosome. If one is to examine a specific individual and find that theyare heterozygous at both markers, such that their two-marker genotype isA₁A₂B₁B₂, then there are two possible configurations: the individual inquestion could have the alleles A₁B₁ on one chromosome and A₂B₂ on theremaining chromosome; alternatively, the individual could have allelesA₁B₂ on one chromosome and A₂B₁ on the other. The arrangement of alleleson a chromosome is called a haplotype. In this illustration, theindividual could have haplotypes A₁B₁/A₂B₂ or A₁B₂/A₂B₁ (see Hartl andClark (1989) for a more complete description). The concept of linkageequilibrium relates the frequency of haplotypes to the allelefrequencies.

Assume that a sample of individuals is selected from a largerpopulation. Considering the two markers described above, each having twoalleles, there are four possible haplotypes: A₁B₁, A₁B₂, A₂B₁ and A₂B₂.Denote the frequencies of these four haplotypes with the followingnotation.P ₁₁=freq(A ₁ B ₁)  (1)P ₁₂=freq(A ₁ B ₂)  (2)P ₂₁=freq(A ₂ B ₁)  (3)P ₂₂=freq(A ₂ B ₂)  (4)The allele frequencies at the two markers are then the sum of differenthaplotype frequencies, it is straightforward to write down a similar setof equations relating single-marker allele frequencies to two-markerhaplotype frequencies:p ₁=freq(A ₁)=P ₁₁ +P ₁₂  (5)p ₂=freq(A ₂)=P ₂₁ +P ₂₂  (6)q ₁=freq(B ₁)=P ₁₁ +P ₂₁  (7)q ₂=freq(B ₂)=P ₁₂ +P ₂₂  (8)Note that the four haplotype frequencies and the allele frequencies ateach marker must sum to a frequency of 1.P ₁₁ +P ₁₂ +P ₂₁ +P ₂₂=1  (9)p ₁ +p ₂=1  (10)q ₁ +q ₂=1  (11)If there is no correlation between the alleles at the two markers, onewould expect that the frequency of the haplotypes would be approximatelythe product of the composite alleles. Therefore,P ₁₁ ≈p ₁ q ₁  (12)P ₁₂ ≈p ₁ q ₂  (13)P ₂₁ ≈p ₂ q ₁  (14)P ₂₂ ≈p ₂ q ₂  (15)These approximating equations (12)-(15) represent the concept of linkageequilibrium where there is independent assortment between the twomarkers—the alleles at the two markers occur together at random. Theseare represented as approximations because linkage equilibrium andlinkage disequilibrium are concepts typically thought of as propertiesof a sample of chromosomes; and as such they are susceptible tostochastic fluctuations due to the sampling process. Empirically, manypairs of genetic markers will be in linkage equilibrium, but certainlynot all pairs.

Having established the concept of linkage equilibrium above, applicantscan now describe the concept of linkage disequilibrium (LD), which isthe deviation from linkage equilibrium. Since the frequency of the A₁B₁haplotype is approximately the product of the allele frequencies for A₁and B₁ under the assumption of linkage equilibrium as statedmathematically in (12), a simple measure for the amount of departurefrom linkage equilibrium is the difference in these two quantities, D,D=P ₁₁ −p ₁ q ₁  (16)D=0 indicates perfect linkage equilibrium. Substantial departures fromD=0 indicates LD in the sample of chromosomes examined. Many propertiesof D are discussed in Lewontin (1964) including the maximum and minimumvalues that D can take. Mathematically, using basic algebra, it can beshown that D can also be written solely in terms of haplotypes:D=P ₁₁ P ₂₂ −P ₁₂ P ₂₁  (17)If one transforms D by squaring it and subsequently dividing by theproduct of the allele frequencies of A₁, A₂, B₁ and B₂, the resultingquantity, called r², is equivalent to the square of the Pearson'scorrelation coefficient commonly used in statistics (e.g. Hoel, 1954).$\begin{matrix}{r^{2} = \frac{D^{2}}{p_{1}p_{2}q_{1}q_{2}}} & (18)\end{matrix}$

As with D, values of r² close to 0 indicate linkage equilibrium betweenthe two markers examined in the sample set. As values of r² increase,the two markers are said to be in linkage disequilibrium. The range ofvalues that r² can take are from 0 to 1. r²=1 when there is a perfectcorrelation between the alleles at the two markers.

In addition, the quantities discussed above are sample-specific. And assuch, it is necessary to formulate notation specific to the samplesstudied. In the approach discussed here, three types of samples are ofprimary interest: (i) a sample of chromosomes from individuals affectedby a disease-related phenotype (cases), (ii) a sample of chromosomesobtained from individuals not affected by the disease-related phenotype(controls), and (iii) a standard sample set used for the construction ofhaplotypes and calculation pairwise linkage disequilibrium. For theallele frequencies used in the development of the method describedbelow, an additional subscript will be added to denote either the caseor control sample sets.p _(1,cs)=freq(A ₁ in cases)  (19)p _(2,cs)=freq(A ₂ in cases)  (20)q _(1,cs)=freq(B ₁ in cases)  (21)q _(2,cs)=freq(B ₂ in cases)  (22)Similarly,p _(1,cs)=freq(A ₁ in controls)  (23)p _(2,cs)=freq(A ₂ in controls)  (24)q _(1,cs)=freq(B ₁ in controls)  (25)q _(2,cs)=freq(B ₂ in controls)  (26)

As a well-accepted sample set is necessary for robust linkagedisequilibrium calculations, data obtained from the International HapMapproject (The International HapMap Consortium 2003, 2005; Thorisson etal, 2005; McVean et al, 2005) can be used for the calculation ofpairwise r² values. Indeed, the samples genotyped for the InternationalHapMap Project were selected to be representative examples from varioushuman sub-populations with sufficient numbers of chromosomes examined todraw meaningful and robust conclusions from the patterns of geneticvariation observed. The International HapMap project website(hapmap.org) contains a description of the project, methods utilized andsamples examined. It is useful to examine empirical data to get a senseof the patterns present in such data.

Haplotype frequencies were explicit arguments in equation (18) above.However, knowing the 2-marker haplotype frequencies requires that phaseto be determined for doubly heterozygous samples. When phase is unknownin the data examined, various algorithms can be used to infer phase fromthe genotype data. This issue was discussed earlier where the doublyheterozygous individual with a 2-SNP genotype of A₁A₂B₁B₂ could have oneof two different sets of chromosomes: A₁B₁/A₂B₂ or A₁B₂/A₂B₁. One suchalgorithm to estimate haplotype frequencies is theexpectation-maximization (EM) algorithm first formalized by Dempster etal (1977). This algorithm is often used in genetics to infer haplotypefrequencies from genotype data (e.g. Excoffier and Slatkin, 1995;Tregouet et al, 2004). It should be noted that for the two-SNP caseexplored here, EM algorithms have very little error provided that theallele frequencies and sample sizes are not too small. The impact on r²values is typically negligible.

As correlated genetic markers share information, interrogation of SNPmarkers in LD with a disease-associated SNP marker can also havesufficient power to detect disease association (Long and Langley, 1999).The relationship between the power to directly find disease-associatedalleles and the power to indirectly detect disease-association wasinvestigated by Pritchard and Przeworski (2001). In a straight-forwardderivation, it can be shown that the power to detect disease associationindirectly at a marker locus in linkage disequilibrium with adisease-association locus is approximately the same as the power todetect disease-association directly at the disease-association locus ifthe sample size is increased by a factor of $\frac{1}{r^{2}}$(the reciprocal of equation 18) at the marker in comparison with thedisease-association locus.

Therefore, if one calculated the power to detect disease-associationindirectly with an experiment having N samples, then equivalent power todirectly detect disease-association (at the actualdisease-susceptibility locus) would necessitate an experiment usingapproximately r²N samples. This elementary relationship between power,sample size and linkage disequilibrium can be used to derive an r²threshold value useful in determining whether or not genotyping markersin linkage disequilibrium with a SNP marker directly associated withdisease status has enough power to indirectly detectdisease-association.

To commence a derivation of the power to detect disease-associatedmarkers through an indirect process, define the effective chromosomalsample size as $\begin{matrix}{{n = \frac{4N_{cs}N_{ct}}{N_{cs} + N_{ct}}};} & (27)\end{matrix}$where N_(cs) and N_(ct) are the numbers of diploid cases and controls,respectively. This is necessary to handle situations where the numbersof cases and controls are not equivalent. For equal case and controlsample sizes, N_(cs)=N_(ct)=N, the value of the effective number ofchromosomes is simply n=2N—as expected. Let power be calculated for asignificance level α (such that traditional P-values below α will bedeemed statistically significant). Define the standard Gaussiandistribution function as Φ(•). Mathematically, $\begin{matrix}{{\Phi(x)} = {\frac{1}{\sqrt{2\quad\pi}}{\int_{- \infty}^{x}{{\mathbb{e}}^{- \frac{\theta^{2}}{2}}{\mathbb{d}\theta}}}}} & (28)\end{matrix}$Alternatively, the following error function notation (Erf) may also beused, $\begin{matrix}{{\Phi(x)} = {\frac{1}{2}\left\lbrack {1 + {{Erf}\left( \frac{x}{\sqrt{2}} \right)}} \right\rbrack}} & (29)\end{matrix}$

For example, Φ(1.644854)=0.95. The value of r² may be derived to yield apre-specified minimum amount of power to detect disease associationthough indirect interrogation. Noting that the LD SNP marker could bethe one that is carrying the disease-association allele, therefore thatthis approach constitutes a lower-bound model where all indirect powerresults are expected to be at least as large as those interrogated.

Denote by β the error rate for not detecting truly disease-associatedmarkers. Therefore, 1−β is the classical definition of statisticalpower. Substituting the Pritchard-Pzreworski result into the samplesize, the power to detect disease association at a significance level ofα is given by the approximation $\begin{matrix}{{{1 - \beta} \cong {\Phi\left\lbrack {\frac{{q_{1,{cs}} - q_{1,{ct}}}}{\sqrt{\frac{{q_{1,{cs}}\left( {1 - q_{1,{cs}}} \right)} + {q_{1,{ct}}\left( {1 - q_{1,{ct}}} \right)}}{r^{2}n}}} - Z_{1 - \frac{\alpha}{2}}} \right\rbrack}};} & (30)\end{matrix}$where Z_(u) is the inverse of the standard normal cumulativedistribution evaluated at u (uε(0,1)). Z_(u)=Φ⁻¹(u), whereΦ(Φ⁻¹(u))=Φ⁻¹(Φ(u))=u. For example, setting α=0.05, and therefore1−α/2=0.975, we obtain Z_(0.975)=1.95996. Next, setting power equal to athreshold of a minimum power of T, $\begin{matrix}{T = {\Phi\left\lbrack {\frac{{q_{1,{cs}} - q_{1,{ct}}}}{\sqrt{\frac{{q_{1,{cs}}\left( {1 - q_{1,{cs}}} \right)} + {q_{1,{ct}}\left( {1 - q_{1,{ct}}} \right)}}{r^{2}n}}} - Z_{1 - {\alpha/2}}} \right\rbrack}} & (31)\end{matrix}$and solving for r², the following threshold r² is obtained:$\begin{matrix}{{r_{T}^{2} - {\frac{\left\lfloor {{q_{1,{cs}}\left( {1 - q_{1,{cs}}} \right)} + {q_{1,{ct}}\left( {1 - q_{1,{ct}}} \right)}} \right\rfloor}{{n\left( {q_{1,{cs}} - q_{1,{ct}}} \right)}^{2}}\left\lbrack {{\Phi^{- 1}(T)} + Z_{1 - \frac{\alpha}{2}}} \right\rbrack}}{{Or},}} & (32) \\{r_{T}^{2} = {\left( \frac{Z_{T} + Z_{1 - \frac{\alpha}{2}}}{n} \right)\left\lbrack \frac{q_{1,{cs}} - \left( q_{1,{cs}} \right)^{2} + q_{1,{ct}} - \left( q_{1,{ct}} \right)^{2}}{\left( {q_{1,{cs}} - q_{1,{ct}}} \right)^{2}} \right\rbrack}} & (33)\end{matrix}$

Suppose that r² is calculated between an interrogated SNP and a numberof other SNPs with varying levels of LD with the interrogated SNP. Thethreshold value r_(T) ² is the minimum value of linkage disequilibriumbetween the interrogated SNP and the potential LD SNPs such that the LDSNP still retains a power greater or equal to T for detectingdisease-association. For example, suppose that SNP rs200 is genotyped ina case-control disease-association study and it is found to beassociated with a disease phenotype. Further suppose that the minorallele frequency in 1,000 case chromosomes was found to be 16% incontrast with a minor allele frequency of 10% in 1,000 controlchromosomes. Given those measurements one could have predicted, prior tothe experiment, that the power to detect disease association at asignificance level of 0.05 was quite high—approximately 98% using a testof allelic association. Applying equation (32) one can calculate aminimum value of r² to indirectly assess disease association assumingthat the minor allele at SNP rs200 is truly disease-predisposing for athreshold level of power. If one sets the threshold level of power to be80%, then r_(T) ²=0.489 given the same significance level and chromosomenumbers as above. Hence, any SNP with a pairwise r² value with rs200greater than 0.489 is expected to have greater than 80% power to detectthe disease association. Further, this is assuming the conservativemodel where the LD SNP is disease-associated only through linkagedisequilibrium with the interrogated SNP rs200.

The contribution or association of particular SNPs and/or SNP haplotypeswith disease phenotypes, such as CHD, enables the SNPs of the presentinvention to be used to develop superior diagnostic tests capable ofidentifying individuals who express a detectable trait, such as CHD, asthe result of a specific genotype, or individuals whose genotype placesthem at an increased or decreased risk of developing a detectable traitat a subsequent time as compared to individuals who do not have thatgenotype. As described herein, diagnostics may be based on a single SNPor a group of SNPs. Combined detection of a plurality of SNPs (forexample, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 24, 25, 30, 32, 48, 50, 64, 96, 100, or any other number in-between,or more, of the SNPs provided in Table 1 and/or Table 2) typicallyincreases the probability of an accurate diagnosis. For example, thepresence of a single SNP known to correlate with CHD might indicate aprobability of 20% that an individual has or is at risk of developingCHD, whereas detection of five SNPs, each of which correlates with CHD,might indicate a probability of 80% that an individual has or is at riskof developing CHD. To further increase the accuracy of diagnosis orpredisposition screening, analysis of the SNPs of the present inventioncan be combined with that of other polymorphisms or other risk factorsof CHD, such as disease symptoms, pathological characteristics, familyhistory, diet, environmental factors or lifestyle factors.

It will, of course, be understood by practitioners skilled in thetreatment or diagnosis of CHD that the present invention generally doesnot intend to provide an absolute identification of individuals who areat risk (or less at risk) of developing CHD, and/or pathologies relatedto CHD, but rather to indicate a certain increased (or decreased) degreeor likelihood of developing the disease based on statisticallysignificant association results. However, this information is extremelyvaluable as it can be used to, for example, initiate preventivetreatments or to allow an individual carrying one or more significantSNPs or SNP haplotypes to foresee warning signs such as minor clinicalsymptoms, or to have regularly scheduled physical exams to monitor forappearance of a condition in order to identify and begin treatment ofthe condition at an early stage. Particularly with diseases that areextremely debilitating or fatal if not treated on time, the knowledge ofa potential predisposition, even if this predisposition is not absolute,would likely contribute in a very significant manner to treatmentefficacy.

The diagnostic techniques of the present invention may employ a varietyof methodologies to determine whether a test subject has a SNP or a SNPpattern associated with an increased or decreased risk of developing adetectable trait or whether the individual suffers from a detectabletrait as a result of a particular polymorphism/mutation, including, forexample, methods which enable the analysis of individual chromosomes forhaplotyping, family studies, single sperm DNA analysis, or somatichybrids. The trait analyzed using the diagnostics of the invention maybe any detectable trait that is commonly observed in pathologies anddisorders related to stenosis or MI.

Another aspect of the present invention relates to a method ofdetermining whether an individual is at risk (or less at risk) ofdeveloping one or more traits or whether an individual expresses one ormore traits as a consequence of possessing a particular trait-causing ortrait-influencing allele. These methods generally involve obtaining anucleic acid sample from an individual and assaying the nucleic acidsample to determine which nucleotide(s) is/are present at one or moreSNP positions, wherein the assayed nucleotide(s) is/are indicative of anincreased or decreased risk of developing the trait or indicative thatthe individual expresses the trait as a result of possessing aparticular trait-causing or trait-influencing allele.

In another embodiment, the SNP detection reagents of the presentinvention are used to determine whether an individual has one or moreSNP allele(s) affecting the level (e.g., the concentration of mRNA orprotein in a sample, etc.) or pattern (e.g., the kinetics of expression,rate of decomposition, stability profile, Km, Vmax, etc.) of geneexpression (collectively, the “gene response” of a cell or bodilyfluid). Such a determination can be accomplished by screening for mRNAor protein expression (e.g., by using nucleic acid arrays, RT-PCR,TaqMan assays, or mass spectrometry), identifying genes having alteredexpression in an individual, genotyping SNPs disclosed in Table 1 and/orTable 2 that could affect the expression of the genes having alteredexpression (e.g., SNPs that are in and/or around the gene(s) havingaltered expression, SNPs in regulatory/control regions, SNPs in and/oraround other genes that are involved in pathways that could affect theexpression of the gene(s) having altered expression, or all SNPs couldbe genotyped), and correlating SNP genotypes with altered geneexpression. In this manner, specific SNP alleles at particular SNP sitescan be identified that affect gene expression.

Pharmacogenomics and Therapeutics/Drug Development

The present invention provides methods for assessing thepharmacogenomics of a subject harboring particular SNP alleles orhaplotypes to a particular therapeutic agent or pharmaceutical compound,or to a class of such compounds. Pharmacogenomics deals with the roleswhich clinically significant hereditary variations (e.g., SNPs) play inthe response to drugs due to altered drug disposition and/or abnormalaction in affected persons. See, e.g., Roses, Nature 405, 857-865(2000); Gould Rothberg, Nature Biotechnology 19, 209-211 (2001);Eichelbaum, Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996); andLinder, Clin. Chem. 43(2):254-266 (1997). The clinical outcomes of thesevariations can result in severe toxicity of therapeutic drugs in certainindividuals or therapeutic failure of drugs in certain individuals as aresult of individual variation in metabolism. Thus, the SNP genotype ofan individual can determine the way a therapeutic compound acts on thebody or the way the body metabolizes the compound. For example, SNPs indrug metabolizing enzymes can affect the activity of these enzymes,which in turn can affect both the intensity and duration of drug action,as well as drug metabolism and clearance.

The discovery of SNPs in drug metabolizing enzymes, drug transporters,proteins for pharmaceutical agents, and other drug targets has explainedwhy some patients do not obtain the expected drug effects, show anexaggerated drug effect, or experience serious toxicity from standarddrug dosages. SNPs can be expressed in the phenotype of the extensivemetabolizer and in the phenotype of the poor metabolizer. Accordingly,SNPs may lead to allelic variants of a protein in which one or more ofthe protein functions in one population are different from those inanother population. SNPs and the encoded variant peptides thus providetargets to ascertain a genetic predisposition that can affect treatmentmodality. For example, in a ligand-based treatment, SNPs may give riseto amino terminal extracellular domains and/or other ligand-bindingregions of a receptor that are more or less active in ligand binding,thereby affecting subsequent protein activation. Accordingly, liganddosage would necessarily be modified to maximize the therapeutic effectwithin a given population containing particular SNP alleles orhaplotypes.

As an alternative to genotyping, specific variant proteins containingvariant amino acid sequences encoded by alternative SNP alleles could beidentified. Thus, pharmacogenomic characterization of an individualpermits the selection of effective compounds and effective dosages ofsuch compounds for prophylactic or therapeutic uses based on theindividual's SNP genotype, thereby enhancing and optimizing theeffectiveness of the therapy. Furthermore, the production of recombinantcells and transgenic animals containing particular SNPs/haplotypes alloweffective clinical design and testing of treatment compounds and dosageregimens. For example, transgenic animals can be produced that differonly in specific SNP alleles in a gene that is orthologous to a humandisease susceptibility gene.

Pharmacogenomic uses of the SNPs of the present invention provideseveral significant advantages for patient care, particularly intreating stenosis or MI. Pharmacogenomic characterization of anindividual, based on an individual's SNP genotype, can identify thoseindividuals unlikely to respond to treatment with a particularmedication and thereby allows physicians to avoid prescribing theineffective medication to those individuals. On the other hand, SNPgenotyping of an individual may enable physicians to select theappropriate medication and dosage regimen that will be most effectivebased on an individual's SNP genotype. This information increases aphysician's confidence in prescribing medications and motivates patientsto comply with their drug regimens. Furthermore, pharmacogenomics mayidentify patients predisposed to toxicity and adverse reactions toparticular drugs or drug dosages. Adverse drug reactions lead to morethan 100,000 avoidable deaths per year in the United States alone andtherefore represent a significant cause of hospitalization and death, aswell as a significant economic burden on the healthcare system (Pfostet. al., Trends in Biotechnology, August 2000.). Thus, pharmacogenomicsbased on the SNPs disclosed herein has the potential to both save livesand reduce healthcare costs substantially.

Pharmacogenomics in general is discussed further in Rose et al.,“Pharmacogenetic analysis of clinically relevant genetic polymorphisms”,Methods Mol Med. 2003; 85:225-37. Pharmacogenomics as it relates toAlzheimer's disease and other neurodegenerative disorders is discussedin Cacabelos, “Pharmacogenomics for the treatment of dementia”, Ann Med.2002; 34(5):357-79, Maimone et al., “Pharmacogenomics ofneurodegenerative diseases”, Eur J Pharmacol. 2001 Feb. 9; 413(1):11-29,and Poirier, “Apolipoprotein E: a pharmacogenetic target for thetreatment of Alzheimer's disease”, Mol Diagn. 1999 December;4(4):335-41. Pharmacogenomics as it relates to cardiovascular disordersis discussed in Siest et al., “Pharmacogenomics of drugs affecting thecardiovascular system”, Clin Chem Lab Med. 2003 April; 41(4):590-9,Mukherjee et al., “Pharmacogenomics in cardiovascular diseases”, ProgCardiovasc Dis. 2002 May-June; 44(6):479-98, and Mooser et al.,“Cardiovascular pharmacogenetics in the SNP era”, J Thromb Haemost. 2003July; 1(7):1398-402. Pharmacogenomics as it relates to cancer isdiscussed in McLeod et al., “Cancer pharmacogenomics: SNPs, chips, andthe individual patient”, Cancer Invest. 2003; 21(4):630-40 and Watterset al., “Cancer pharmacogenomics: current and future applications”,Biochim Biophys Acta. 2003 Mar. 17; 1603(2):99-111.

The SNPs of the present invention also can be used to identify noveltherapeutic targets for MI. For example, genes containing thedisease-associated variants (“variant genes”) or their products, as wellas genes or their products that are directly or indirectly regulated byor interacting with these variant genes or their products, can betargeted for the development of therapeutics that, for example, treatthe disease or prevent or delay disease onset. The therapeutics may becomposed of, for example, small molecules, proteins, protein fragmentsor peptides, antibodies, nucleic acids, or their derivatives or mimeticswhich modulate the functions or levels of the target genes or geneproducts.

The SNP-containing nucleic acid molecules disclosed herein, and theircomplementary nucleic acid molecules, may be used as antisenseconstructs to control gene expression in cells, tissues, and organisms.Antisense technology is well established in the art and extensivelyreviewed in Antisense Drug Technology: Principles, Strategies, andApplications, Crooke (ed.), Marcel Dekker, Inc.: New York (2001). Anantisense nucleic acid molecule is generally designed to becomplementary to a region of mRNA expressed by a gene so that theantisense molecule hybridizes to the mRNA and thereby blocks translationof mRNA into protein. Various classes of antisense oligonucleotides areused in the art, two of which are cleavers and blockers. Cleavers, bybinding to target RNAs, activate intracellular nucleases (e.g., RNaseHor RNase L) that cleave the target RNA. Blockers, which also bind totarget RNAs, inhibit protein translation through steric hindrance ofribosomes. Exemplary blockers include peptide nucleic acids,morpholinos, locked nucleic acids, and methylphosphonates (see, e.g.,Thompson, Drug Discovery Today, 7 (17): 912-917 (2002)). Antisenseoligonucleotides are directly useful as therapeutic agents, and are alsouseful for determining and validating gene function (e.g., in geneknock-out or knock-down experiments).

Antisense technology is further reviewed in: Lavery et al., “Antisenseand RNAi: powerful tools in drug target discovery and validation”, CurrOpin Drug Discov Devel. 2003 July; 6(4):561-9; Stephens et al.,“Antisense oligonucleotide therapy in cancer”, Curr Opin Mol Ther. 2003April; 5(2): 118-22; Kurreck, “Antisense technologies. Improvementthrough novel chemical modifications”, Eur J. Biochem. 2003 April;270(8): 1628-44; Dias et al., “Antisense oligonucleotides: basicconcepts and mechanisms”, Mol Cancer Ther. 2002 March; 1(5):347-55;Chen, “Clinical development of antisense oligonucleotides as anti-cancertherapeutics”, Methods Mol Med. 2003; 75:621-36; Wang et al., “Antisenseanticancer oligonucleotide therapeutics”, Curr Cancer Drug Targets. 2001November; 1(3):177-96; and Bennett, “Efficiency of antisenseoligonucleotide drug discovery”, Antisense Nucleic Acid Drug Dev. 2002June; 12(3):215-24.

The SNPs of the present invention are particularly useful for designingantisense reagents that are specific for particular nucleic acidvariants. Based on the SNP information disclosed herein, antisenseoligonucleotides can be produced that specifically target mRNA moleculesthat contain one or more particular SNP nucleotides. In this manner,expression of mRNA molecules that contain one or more undesiredpolymorphisms (e.g., SNP nucleotides that lead to a defective proteinsuch as an amino acid substitution in a catalytic domain) can beinhibited or completely blocked. Thus, antisense oligonucleotides can beused to specifically bind a particular polymorphic form (e.g., a SNPallele that encodes a defective protein), thereby inhibiting translationof this form, but which do not bind an alternative polymorphic form(e.g., an alternative SNP nucleotide that encodes a protein havingnormal function):

Antisense molecules can be used to inactivate mRNA in order to inhibitgene expression and production of defective proteins. Accordingly, thesemolecules can be used to treat a disorder, such as stenosis or MI,characterized by abnormal or undesired gene expression or expression ofcertain defective proteins. This technique can involve cleavage by meansof ribozymes containing nucleotide sequences complementary to one ormore regions in the mRNA that attenuate the ability of the mRNA to betranslated. Possible mRNA regions include, for example, protein-codingregions and particularly protein-coding regions corresponding tocatalytic activities, substrate/ligand binding, or other functionalactivities of a protein.

The SNPs of the present invention are also useful for designing RNAinterference reagents that specifically target nucleic acid moleculeshaving particular SNP variants. RNA interference (RNAi), also referredto as gene silencing, is based on using double-stranded RNA (dsRNA)molecules to turn genes off. When introduced into a cell, dsRNAs areprocessed by the cell into short fragments (generally about 21, 22, or23 nucleotides in length) known as small interfering RNAs (siRNAs) whichthe cell uses in a sequence-specific manner to recognize and destroycomplementary RNAs (Thompson, Drug Discovery Today, 7 (17): 912-917(2002)). Accordingly, an aspect of the present invention specificallycontemplates isolated nucleic acid molecules that are about 18-26nucleotides in length, preferably 19-25 nucleotides in length, and morepreferably 20, 21, 22, or 23 nucleotides in length, and the use of thesenucleic acid molecules for RNAi. Because RNAi molecules, includingsiRNAs, act in a sequence-specific manner, the SNPs of the presentinvention can be used to design RNAi reagents that recognize and destroynucleic acid molecules having specific SNP alleles/nucleotides (such asdeleterious alleles that lead to the production of defective proteins),while not affecting nucleic acid molecules having alternative SNPalleles (such as alleles that encode proteins having normal function).As with antisense reagents, RNAi reagents may be directly useful astherapeutic agents (e.g., for turning off defective, disease-causinggenes), and are also useful for characterizing and validating genefunction (e.g., in gene knock-out or knock-down experiments).

The following references provide a further review of RNAi: Reynolds etal., “Rational siRNA design for RNA interference”, Nat Biotechnol. 2004March; 22(3):326-30. Epub 2004 Feb. 1; Chi et al., “Genomewide view ofgene silencing by small interfering RNAs”, PNAS 100(11):6343-6346, 2003;Vickers et al., “Efficient Reduction of Target RNAs by Small InterferingRNA and RNase H-dependent Antisense Agents”, J. Biol. Chem. 278:7108-7118, 2003; Agami, “RNAi and related mechanisms and their potentialuse for therapy”, Curr Opin Chem Biol. 2002 December; 6(6):829-34;Lavery et al., “Antisense and RNAi: powerful tools in drug targetdiscovery and validation”, Curr Opin Drug Discov Devel. 2003 July;6(4):561-9; Shi, “Mammalian RNAi for the masses”, Trends Genet 2003January; 19(1):9-12), Shuey et al., “RNAi: gene-silencing in therapeuticintervention”, Drug Discovery Today 2002 October; 7(20): 1040-1046;McManus et al., Nat Rev Genet 2002 October; 3(10):737-47; Xia et al.,Nat Biotechnol 2002 October; 20(10):1006-10; Plasterk et al., Curr OpinGenet Dev 2000 October; 10(5):562-7; Bosher et al., Nat Cell Biol 2000February; 2(2):E31-6; and Hunter, Curr Biol 1999 Jun. 17; 9(12):R440-2).

A subject suffering from a pathological condition, such as stenosis orMI, ascribed to a SNP may be treated so as to correct the genetic defect(see Kren et al., Proc. Natl. Acad. Sci. USA 96:10349-10354 (1999)).Such a subject can be identified by any method that can detect thepolymorphism in a biological sample drawn from the subject. Such agenetic defect may be permanently corrected by administering to such asubject a nucleic acid fragment incorporating a repair sequence thatsupplies the normal/wild-type nucleotide at the position of the SNP.This site-specific repair sequence can encompass an RNA/DNAoligonucleotide that operates to promote endogenous repair of asubject's genomic DNA. The site-specific repair sequence is administeredin an appropriate vehicle, such as a complex with polyethylenimine,encapsulated in anionic liposomes, a viral vector such as an adenovirus,or other pharmaceutical composition that promotes intracellular uptakeof the administered nucleic acid. A genetic defect leading to an inbornpathology may then be overcome, as the chimeric oligonucleotides induceincorporation of the normal sequence into the subject's genome. Uponincorporation, the normal gene product is expressed, and the replacementis propagated, thereby engendering a permanent repair and therapeuticenhancement of the clinical condition of the subject.

In cases in which a cSNP results in a variant protein that is ascribedto be the cause of, or a contributing factor to, a pathologicalcondition, a method of treating such a condition can includeadministering to a subject experiencing the pathology thewild-type/normal cognate of the variant protein. Once administered in aneffective dosing regimen, the wild-type cognate provides complementationor remediation of the pathological condition.

The invention further provides a method for identifying a compound oragent that can be used to treat stenosis or MI. The SNPs disclosedherein are useful as targets for the identification and/or developmentof therapeutic agents. A method for identifying a therapeutic agent orcompound typically includes assaying the ability of the agent orcompound to modulate the activity and/or expression of a SNP-containingnucleic acid or the encoded product and thus identifying an agent or acompound that can be used to treat a disorder characterized by undesiredactivity or expression of the SNP-containing nucleic acid or the encodedproduct. The assays can be performed in cell-based and cell-freesystems. Cell-based assays can include cells naturally expressing thenucleic acid molecules of interest or recombinant cells geneticallyengineered to express certain nucleic acid molecules.

Variant gene expression in a stenosis or MI patient can include, forexample, either expression of a SNP-containing nucleic acid sequence(for instance, a gene that contains a SNP can be transcribed into anmRNA transcript molecule containing the SNP, which can in turn betranslated into a variant protein) or altered expression of anormal/wild-type nucleic acid sequence due to one or more SNPs (forinstance, a regulatory/control region can contain a SNP that affects thelevel or pattern of expression of a normal transcript).

Assays for variant gene expression can involve direct assays of nucleicacid levels (e.g., mRNA levels), expressed protein levels, or ofcollateral compounds involved in a signal pathway. Further, theexpression of genes that are up- or down-regulated in response to thesignal pathway can also be assayed. In this embodiment, the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

Modulators of variant gene expression can be identified in a methodwherein, for example, a cell is contacted with a candidatecompound/agent and the expression of mRNA determined. The level ofexpression of mRNA in the presence of the candidate compound is comparedto the level of expression of mRNA in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof variant gene expression based on this comparison and be used to treata disorder such as stenosis or MI that is characterized by variant geneexpression (e.g., either expression of a SNP-containing nucleic acid oraltered expression of a normal/wild-type nucleic acid molecule due toone or more SNPs that affect expression of the nucleic acid molecule)due to one or more SNPs of the present invention. When expression ofmRNA is statistically significantly greater in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of nucleic acid expression. When nucleic acidexpression is statistically significantly less in the presence of thecandidate compound than in its absence, the candidate compound isidentified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the SNP orassociated nucleic acid domain (e.g., catalytic domain,ligand/substrate-binding domain, regulatory/control region, etc.) orgene, or the encoded mRNA transcript, as a target, using a compoundidentified through drug screening as a gene modulator to modulatevariant nucleic acid expression. Modulation can include eitherup-regulation (i.e., activation or agonization) or down-regulation(i.e., suppression or antagonization) of nucleic acid expression.

Expression of mRNA transcripts and encoded proteins, either wild type orvariant, may be altered in individuals with a particular SNP allele in aregulatory/control element, such as a promoter or transcription factorbinding domain, that regulates expression. In this situation, methods oftreatment and compounds can be identified, as discussed herein, thatregulate or overcome the variant regulatory/control element, therebygenerating normal, or healthy, expression levels of either the wild typeor variant protein.

The SNP-containing nucleic acid molecules of the present invention arealso useful for monitoring the effectiveness of modulating compounds onthe expression or activity of a variant gene, or encoded product, inclinical trials or in a treatment regimen. Thus, the gene expressionpattern can serve as an indicator for the continuing effectiveness oftreatment with the compound, particularly with compounds to which apatient can develop resistance, as well as an indicator for toxicities.The gene expression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

In another aspect of the present invention, there is provided apharmaceutical pack comprising a therapeutic agent (e.g., a smallmolecule drug, antibody, peptide, antisense or RNAi nucleic acidmolecule, etc.) and a set of instructions for administration of thetherapeutic agent to humans diagnostically tested for one or more SNPsor SNP haplotypes provided by the present invention.

The SNPs/haplotypes of the present invention are also useful forimproving many different aspects of the drug development process. Forinstance, an aspect of the present invention includes selectingindividuals for clinical trials based on their SNP genotype. Forexample, individuals with SNP genotypes that indicate that they arelikely to positively respond to a drug can be included in the trials,whereas those individuals whose SNP genotypes indicate that they areless likely to or would not respond to the drug, or who are at risk forsuffering toxic effects or other adverse reactions, can be excluded fromthe clinical trials. This not only can improve the safety of clinicaltrials, but also can enhance the chances that the trial will demonstratestatistically significant efficacy. Furthermore, the SNPs of the presentinvention may explain why certain previously developed drugs performedpoorly in clinical trials and may help identify a subset of thepopulation that would benefit from a drug that had previously performedpoorly in clinical trials, thereby “rescuing” previously developeddrugs, and enabling the drug to be made available to a particularstenosis or MI patient population that can benefit from it.

SNPs have many important uses in drug discovery, screening, anddevelopment. A high probability exists that, for any gene/proteinselected as a potential drug target, variants of that gene/protein willexist in a patient population. Thus, determining the impact ofgene/protein variants on the selection and delivery of a therapeuticagent should be an integral aspect of the drug discovery and developmentprocess. (Jazwinska, A Trends Guide to Genetic Variation and GenomicMedicine, 2002 March; S30-S36).

Knowledge of variants (e.g., SNPs and any corresponding amino acidpolymorphisms) of a particular therapeutic target (e.g., a gene, mRNAtranscript, or protein) enables parallel screening of the variants inorder to identify therapeutic candidates (e.g., small moleculecompounds, antibodies, antisense or RNAi nucleic acid compounds, etc.)that demonstrate efficacy across variants (Rothberg, Nat Biotechnol 2001March; 19(3):209-11). Such therapeutic candidates Would be expected toshow equal efficacy across a larger segment of the patient population,thereby leading to a larger potential market for the therapeuticcandidate.

Furthermore, identifying variants of a potential therapeutic targetenables the most common form of the target to be used for selection oftherapeutic candidates, thereby helping to ensure that the experimentalactivity that is observed for the selected candidates reflects the realactivity expected in the largest proportion of a patient population(Jazwinska, A Trends Guide to Genetic Variation and Genomic Medicine,2002 March; S30-S36).

Additionally, screening therapeutic candidates against all knownvariants of a target can enable the early identification of potentialtoxicities and adverse reactions relating to particular variants. Forexample, variability in drug absorption, distribution, metabolism andexcretion (ADME) caused by, for example, SNPs in therapeutic targets ordrug metabolizing genes, can be identified, and this information can beutilized during the drug development process to minimize variability indrug disposition and develop therapeutic agents that are safer across awider range of a patient population. The SNPs of the present invention,including the variant proteins and encoding polymorphic nucleic acidmolecules provided in Tables 1-2, are useful in conjunction with avariety of toxicology methods established in the art, such as those setforth in Current Protocols in Toxicology, John Wiley & Sons, Inc., N.Y.

Furthermore, therapeutic agents that target any art-known proteins (ornucleic acid molecules, either RNA or DNA) may cross-react with thevariant proteins (or polymorphic nucleic acid molecules) disclosed inTable 1, thereby significantly affecting the pharmacokinetic propertiesof the drug. Consequently, the protein variants and the SNP-containingnucleic acid molecules disclosed in Tables 1-2 are useful in developing,screening, and evaluating therapeutic agents that target correspondingart-known protein forms (or nucleic acid molecules). Additionally, asdiscussed above, knowledge of all polymorphic forms of a particular drugtarget enables the design of therapeutic agents that are effectiveagainst most or all such polymorphic forms of the drug target.

Pharmaceutical Compositions and Administration Thereof

Any of the MI-associated proteins, and encoding nucleic acid molecules,disclosed herein can be used as therapeutic targets (or directly usedthemselves as therapeutic compounds) for treating stenosis or MI andrelated pathologies, and the present disclosure enables therapeuticcompounds (e.g., small molecules, antibodies, therapeutic proteins, RNAiand antisense molecules, etc.) to be developed that target (or arecomprised of) any of these therapeutic targets.

In general, a therapeutic compound will be administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. The actualamount of the therapeutic compound of this invention, i.e., the activeingredient, will depend upon numerous factors such as the severity ofthe disease to be treated, the age and relative health of the subject,the potency of the compound used, the route and form of administration,and other factors.

Therapeutically effective amounts of therapeutic compounds may rangefrom, for example, approximately 0.01-50 mg per kilogram body weight ofthe recipient per day; preferably about 0.1-20 mg/kg/day. Thus, as anexample, for administration to a 70 kg person, the dosage range wouldmost preferably be about 7 mg to 1.4 g per day.

In general, therapeutic compounds will be administered as pharmaceuticalcompositions by any one of the following routes: oral, systemic (e.g.,transdermal, intranasal, or by suppository), or parenteral (e.g.,intramuscular, intravenous, or subcutaneous) administration. Thepreferred manner of administration is oral or parenteral using aconvenient daily dosage regimen, which can be adjusted according to thedegree of affliction. Oral compositions can take the form of tablets,pills, capsules, semisolids, powders, sustained release formulations,solutions, suspensions, elixirs, aerosols, or any other appropriatecompositions.

The choice of formulation depends on various factors such as the mode ofdrug administration (e.g., for oral administration, formulations in theform of tablets, pills, or capsules are preferred) and thebioavailability of the drug substance. Recently, pharmaceuticalformulations have been developed especially for drugs that show poorbioavailability based upon the principle that bioavailability can beincreased by increasing the surface area, i.e., decreasing particlesize. For example, U.S. Pat. No. 4,107,288 describes a pharmaceuticalformulation having particles in the size range from 10 to 1,000 nm inwhich the active material is supported on a cross-linked matrix ofmacromolecules. U.S. Pat. No. 5,145,684 describes the production of apharmaceutical formulation in which the drug substance is pulverized tonanoparticles (average particle size of 400 nm) in the presence of asurface modifier and then dispersed in a liquid medium to give apharmaceutical formulation that exhibits remarkably highbioavailability.

Pharmaceutical compositions are comprised of, in general, a therapeuticcompound in combination with at least one pharmaceutically acceptableexcipient. Acceptable excipients are non-toxic, aid administration, anddo not adversely affect the therapeutic benefit of the therapeuticcompound. Such excipients may be any solid, liquid, semi-solid or, inthe case of an aerosol composition, gaseous excipient that is generallyavailable to one skilled in the art.

Solid pharmaceutical excipients include starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like. Liquid and semisolid excipientsmay be selected from glycerol, propylene glycol, water, ethanol andvarious oils, including those of petroleum, animal, vegetable orsynthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesameoil, etc. Preferred liquid carriers, particularly for injectablesolutions, include water, saline, aqueous dextrose, and glycols.

Compressed gases may be used to disperse a compound of this invention inaerosol form. Inert gases suitable for this purpose are nitrogen, carbondioxide, etc.

Other suitable pharmaceutical excipients and their formulations aredescribed in Remington's Pharmaceutical Sciences, edited by E. W. Martin(Mack Publishing Company, 18^(th) ed., 1990).

The amount of the therapeutic compound in a formulation can vary withinthe full range employed by those skilled in the art. Typically, theformulation will contain, on a weight percent (wt %) basis, from about0.01-99.99 wt % of the therapeutic compound based on the totalformulation, with the balance being one or more suitable pharmaceuticalexcipients. Preferably, the compound is present at a level of about 1-80wt %.

Therapeutic compounds can be administered alone or in combination withother therapeutic compounds or in combination with one or more otheractive ingredient(s). For example, an inhibitor or stimulator of anstenosis or MI-associated protein can be administered in combinationwith another agent that inhibits or stimulates the activity of the sameor a different MI-associated protein to thereby counteract the affectsof stenosis or MI.

For further information regarding pharmacology, see Current Protocols inPharmacology, John Wiley & Sons, Inc., N.Y.

Human Identification Applications

In addition to their diagnostic and therapeutic uses in stenosis or MIand related pathologies, the SNPs provided by the present invention arealso useful as human identification markers for such applications asforensics, paternity testing, and biometrics (see, e.g., Gill, “Anassessment of the utility of single nucleotide polymorphisms (SNPs) forforensic purposes”, Int J Legal Med. 2001; 114(4-5):204-10). Geneticvariations in the nucleic acid sequences between individuals can be usedas genetic markers to identify individuals and to associate a biologicalsample with an individual. Determination of which nucleotides occupy aset of SNP positions in an individual identifies a set of SNP markersthat distinguishes the individual. The more SNP positions that areanalyzed, the lower the probability that the set of SNPs in oneindividual is the same as that in an unrelated individual. Preferably,if multiple sites are analyzed, the sites are unlinked (i.e., inheritedindependently). Thus, preferred sets of SNPs can be selected from amongthe SNPs disclosed herein, which may include SNPs on differentchromosomes, SNPs on different chromosome arms, and/or SNPs that aredispersed over substantial distances along the same chromosome arm.

Furthermore, among the SNPs disclosed herein, preferred SNPs for use incertain forensic/human identification applications include SNPs locatedat degenerate codon positions (i.e., the third position in certaincodons which can be one of two or more alternative nucleotides and stillencode the same amino acid), since these SNPs do not affect the encodedprotein. SNPs that do not affect the encoded protein are expected to beunder less selective pressure and are therefore expected to be morepolymorphic in a population, which is typically an advantage forforensic/human identification applications. However, for certainforensics/human identification applications, such as predictingphenotypic characteristics (e.g., inferring ancestry or inferring one ormore physical characteristics of an individual) from a DNA sample, itmay be desirable to utilize SNPs that affect the encoded protein.

For many of the SNPs disclosed in Tables 1-2 (which are identified as“Applera” SNP source), Tables 1-2 provide SNP allele frequenciesobtained by re-sequencing the DNA of chromosomes from 39 individuals(Tables 1-2 also provide allele frequency information for “Celera”source SNPs and, where available, public SNPs from dbEST, HGBASE, and/orHGMD). The allele frequencies provided in Tables 1-2 enable these SNPsto be readily used for human identification applications. Although anySNP disclosed in Table 1 and/or Table 2 could be used for humanidentification, the closer that the frequency of the minor allele at aparticular SNP site is to 50%, the greater the ability of that SNP todiscriminate between different individuals in a population since itbecomes increasingly likely that two randomly selected individuals wouldhave different alleles at that SNP site. Using the SNP allelefrequencies provided in Tables 1-2, one of ordinary skill in the artcould readily select a subset of SNPs for which the frequency of theminor allele is, for example, at least 1%, 2%, 5%, 10%, 20%, 25%, 30%,40%, 45%, or 50%, or any other frequency in-between. Thus, since Tables1-2 provide allele frequencies based on the re-sequencing of thechromosomes from 39 individuals, a subset of SNPs could readily beselected for human identification in which the total allele count of theminor allele at a particular SNP site is, for example, at least 1, 2, 4,8, 10, 16, 20, 24, 30, 32, 36, 38, 39, 40, or any other numberin-between.

Furthermore, Tables 1-2 also provide population group (interchangeablyreferred to herein as ethnic or racial groups) information coupled withthe extensive allele frequency information. For example, the group of 39individuals whose DNA was re-sequenced was made-up of 20 Caucasians and19 African-Americans. This population group information enables furtherrefinement of SNP selection for human identification. For example,preferred SNPs for human identification can be selected from Tables 1-2that have similar allele frequencies in both the Caucasian andAfrican-American populations; thus, for example, SNPs can be selectedthat have equally high discriminatory power in both populations.Alternatively, SNPs can be selected for which there is a statisticallysignificant difference in allele frequencies between the Caucasian andAfrican-American populations (as an extreme example, a particular allelemay be observed only in either the Caucasian or the African-Americanpopulation group but not observed in the other population group); suchSNPs are useful, for example, for predicting the race/ethnicity of anunknown perpetrator from a biological sample such as a hair or bloodstain recovered at a crime scene. For a discussion of using SNPs topredict ancestry from a DNA sample, including statistical methods, seeFrudakis et al., “A Classifier for the SNP-Based Inference of Ancestry,”Journal of Forensic Sciences 2003; 48(4):771-782.

SNPs have numerous advantages over other types of polymorphic markers,such as short tandem repeats (STRs). For example, SNPs can be easilyscored and are amenable to automation, making SNPs the markers of choicefor large-scale forensic databases. SNPs are found in much greaterabundance throughout the genome than repeat polymorphisms. Populationfrequencies of two polymorphic forms can usually be determined withgreater accuracy than those of multiple polymorphic forms atmulti-allelic loci. SNPs are mutationaly more stable than repeatpolymorphisms. SNPs are not susceptible to artefacts such as stutterbands that can hinder analysis. Stutter bands are frequently encounteredwhen analyzing repeat polymorphisms, and are particularly troublesomewhen analyzing samples such as crime scene samples that may containmixtures of DNA from multiple sources. Another significant advantage ofSNP markers over STR markers is the much shorter length of nucleic acidneeded to score a SNP. For example, STR markers are generally severalhundred base pairs in length. A SNP, on the other hand, comprises asingle nucleotide, and generally a short conserved region on either sideof the SNP position for primer and/or probe binding. This makes SNPsmore amenable to typing in highly degraded or aged biological samplesthat are frequently encountered in forensic casework in which DNA may befragmented into short pieces.

SNPs also are not subject to microvariant and “off-ladder” allelesfrequently encountered when analyzing STR loci. Microvariants aredeletions or insertions within a repeat unit that change the size of theamplified DNA product so that the amplified product does not migrate atthe same rate as reference alleles with normal sized repeat units. Whenseparated by size, such as by electrophoresis on a polyacrylamide gel,microvariants do not align with a reference allelic ladder of standardsized repeat units, but rather migrate between the reference alleles.The reference allelic ladder is used for precise sizing of alleles forallele classification; therefore alleles that do not align with thereference allelic ladder lead to substantial analysis problems.Furthermore, when analyzing multi-allelic repeat polymorphisms,occasionally an allele is found that consists of more or less repeatunits than has been previously seen in the population, or more or lessrepeat alleles than are included in a reference allelic ladder. Thesealleles will migrate outside the size range of known alleles in areference allelic ladder, and therefore are referred to as “off-ladder”alleles. In extreme cases, the allele may contain so few or so manyrepeats that it migrates well out of the range of the reference allelicladder. In this situation, the allele may not even be observed, or, withmultiplex analysis, it may migrate within or close to the size range foranother locus, further confounding analysis.

SNP analysis avoids the problems of microvariants and off-ladder allelesencountered in STR analysis. Importantly, microvariants and off-ladderalleles may provide significant problems, and may be completely missed,when using analysis methods such as oligonucleotide hybridizationarrays, which utilize oligonucleotide probes specific for certain knownalleles. Furthermore, off-ladder alleles and microvariants encounteredwith STR analysis, even when correctly typed, may lead to improperstatistical analysis, since their frequencies in the population aregenerally unknown or poorly characterized, and therefore the statisticalsignificance of a matching genotype may be questionable. All theseadvantages of SNP analysis are considerable in light of the consequencesof most DNA identification cases, which may lead to life imprisonmentfor an individual, or re-association of remains to the family of adeceased individual.

DNA can be isolated from biological samples such as blood, bone, hair,saliva, or semen, and compared with the DNA from a reference source atparticular SNP positions. Multiple SNP markers can be assayedsimultaneously in order to increase the power of discrimination and thestatistical significance of a matching genotype. For example,oligonucleotide arrays can be used to genotype a large number of SNPssimultaneously. The SNPs provided by the present invention can beassayed in combination with other polymorphic genetic markers, such asother SNPs known in the art or STRs, in order to identify an individualor to associate an individual with a particular biological sample.

Furthermore, the SNPs provided by the present invention can be genotypedfor inclusion in a database of DNA genotypes, for example, a criminalDNA databank such as the FBI's Combined DNA Index System (CODIS)database. A genotype obtained from a biological sample of unknown sourcecan then be queried against the database to find a matching genotype,with the SNPs of the present invention providing nucleotide positions atwhich to compare the known and unknown DNA sequences for identity.Accordingly, the present invention provides a database comprising novelSNPs or SNP alleles of the present invention (e.g., the database cancomprise information indicating which alleles are possessed byindividual members of a population at one or more novel SNP sites of thepresent invention), such as for use in forensics, biometrics, or otherhuman identification applications. Such a database typically comprises acomputer-based system in which the SNPs or SNP alleles of the presentinvention are recorded on a computer readable medium (see the section ofthe present specification entitled “Computer-Related Embodiments”).

The SNPs of the present invention can also be assayed for use inpaternity testing. The object of paternity testing is usually todetermine whether a male is the father of a child. In most cases, themother of the child is known and thus, the mother's contribution to thechild's genotype can be traced. Paternity testing investigates whetherthe part of the child's genotype not attributable to the mother isconsistent with that of the putative father. Paternity testing can beperformed by analyzing sets of polymorphisms in the putative father andthe child, with the SNPs of the present invention providing nucleotidepositions at which to compare the putative father's and child's DNAsequences for identity. If the set of polymorphisms in the childattributable to the father does not match the set of polymorphisms ofthe putative father, it can be concluded, barring experimental error,that the putative father is not the father of the child. If the set ofpolymorphisms in the child attributable to the father match the set ofpolymorphisms of the putative father, a statistical calculation can beperformed to determine the probability of coincidental match, and aconclusion drawn as to the likelihood that the putative father is thetrue biological father of the child.

In addition to paternity testing, SNPs are also useful for other typesof kinship testing, such as for verifying familial relationships forimmigration purposes, or for cases in which an individual alleges to berelated to a deceased individual in order to claim an inheritance fromthe deceased individual, etc. For further information regarding theutility of SNPs for paternity testing and other types of kinshiptesting, including methods for statistical analysis, see Krawczak,“Informativity assessment for biallelic single nucleotidepolymorphisms”, Electrophoresis 1999 June; 20(8):1676-81.

The use of the SNPs of the present invention for human identificationfurther extends to various authentication systems, commonly referred toas biometric systems, which typically convert physical characteristicsof humans (or other organisms) into digital data. Biometric systemsinclude various technological devices that measure such uniqueanatomical or physiological characteristics as finger, thumb, or palmprints; hand geometry; vein patterning on the back of the hand; bloodvessel patterning of the retina and color and texture of the iris;facial characteristics; voice patterns; signature and typing dynamics;and DNA. Such physiological measurements can be used to verify identityand, for example, restrict or allow access based on the identification.Examples of applications for biometrics include physical area security,computer and network security, aircraft passenger check-in and boarding,financial transactions, medical records access, government benefitdistribution, voting, law enforcement, passports, visas and immigration,prisons, various military applications, and for restricting access toexpensive or dangerous items, such as automobiles or guns (see, forexample, O'Connor, Stanford Technology Law Review and U.S. Pat. No.6,119,096).

Groups of SNPs, particularly the SNPs provided by the present invention,can be typed to uniquely identify an individual for biometricapplications such as those described above. Such SNP typing can readilybe accomplished using, for example, DNA chips/arrays. Preferably, aminimally invasive means for obtaining a DNA sample is utilized. Forexample, PCR amplification enables sufficient quantities of DNA foranalysis to be obtained from buccal swabs or fingerprints, which containDNA-containing skin cells and oils that are naturally transferred duringcontact.

Further information regarding techniques for using SNPs inforensic/human identification applications can be found in, for example,Current Protocols in Human Genetics, John Wiley & Sons, N.Y. (2002),14.1-14.7.

Variant Proteins, Antibodies, Vectors, Host Cells, & Uses Thereof

Variant Proteins Encoded by SNP-Containing Nucleic Acid Molecules

The present invention provides SNP-containing nucleic acid molecules,many of which encode proteins having variant amino acid sequences ascompared to the art-known (i.e., wild-type) proteins. Amino acidsequences encoded by the polymorphic nucleic acid molecules of thepresent invention are referred to as SEQ ID NOS: 68-134 in Table 1 andprovided in the Sequence Listing. These variants will generally bereferred to herein as variant proteins/peptides/polypeptides, orpolymorphic proteins/peptides/polypeptides of the present invention. Theterms “protein,” “peptide,” and “polypeptide” are used hereininterchangeably.

A variant protein of the present invention may be encoded by, forexample, a nonsynonymous nucleotide substitution at any one of the cSNPpositions disclosed herein. In addition, variant proteins may alsoinclude proteins whose expression, structure, and/or function is alteredby a SNP disclosed herein, such as a SNP that creates or destroys a stopcodon, a SNP that affects splicing, and a SNP in control/regulatoryelements, e.g. promoters, enhancers, or transcription factor bindingdomains.

As used herein, a protein or peptide is said to be “isolated” or“purified” when it is substantially free of cellular material orchemical precursors or other chemicals. The variant proteins of thepresent invention can be purified to homogeneity or other lower degreesof purity. The level of purification will be based on the intended use.The key feature is that the preparation allows for the desired functionof the variant protein, even if in the presence of considerable amountsof other components.

As used herein, “substantially free of cellular material” includespreparations of the variant protein having less than about 30% (by dryweight) other proteins (i.e., contaminating protein), less than about20% other proteins, less than about 10% other proteins, or less thanabout 5% other proteins. When the variant protein is recombinantlyproduced, it can also be substantially free of culture medium, i.e.,culture medium represents less than about 20% of the volume of theprotein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the variant protein in which it isseparated from chemical precursors or other chemicals that are involvedin its synthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thevariant protein having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

An isolated variant protein may be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant host cells), or synthesized using known protein synthesismethods. For example, a nucleic acid molecule containing SNP(s) encodingthe variant protein can be cloned into an expression vector, theexpression vector introduced into a host cell, and the variant proteinexpressed in the host cell. The variant protein can then be isolatedfrom the cells by any appropriate purification scheme using standardprotein purification techniques. Examples of these techniques aredescribed in detail below (Sambrook and Russell, 2000, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

The present invention provides isolated variant proteins that comprise,consist of or consist essentially of amino acid sequences that containone or more variant amino acids encoded by one or more codons thatcontain a SNP of the present invention.

Accordingly, the present invention provides variant proteins thatconsist of amino acid sequences that contain one or more amino acidpolymorphisms (or truncations or extensions due to creation ordestruction of a stop codon, respectively) encoded by the SNPs providedin Table 1 and/or Table 2. A protein consists of an amino acid sequencewhen the amino acid sequence is the entire amino acid sequence of theprotein.

The present invention further provides variant proteins that consistessentially of amino acid sequences that contain one or more amino acidpolymorphisms (or truncations or extensions due to creation ordestruction of a stop codon, respectively) encoded by the SNPs providedin Table 1 and/or Table 2. A protein consists essentially of an aminoacid sequence when such an amino acid sequence is present with only afew additional amino acid residues in the final protein.

The present invention further provides variant proteins that compriseamino acid sequences that contain one or more amino acid polymorphisms(or truncations or extensions due to creation or destruction of a stopcodon, respectively) encoded by the SNPs provided in Table 1 and/orTable 2. A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein may contain only the variantamino acid sequence or have additional amino acid residues, such as acontiguous encoded sequence that is naturally associated with it orheterologous amino acid residues. Such a protein can have a fewadditional amino acid residues or can comprise many more additionalamino acids. A brief description of how various types of these proteinscan be made and isolated is provided below.

The variant proteins of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a variant protein operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the variant protein. “Operatively linked”indicates that the coding sequences for the variant protein and theheterologous protein are ligated in-frame. The heterologous protein canbe fused to the N-terminus or C-terminus of the variant protein. Inanother embodiment, the fusion protein is encoded by a fusionpolynucleotide that is synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and re-amplified to generate a chimeric genesequence (see Ausubel et al., Current Protocols in Molecular Biology,1992). Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST protein). A variantprotein-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the variantprotein.

In many uses, the fusion protein does not affect the activity of thevariant protein. The fusion protein can include, but is not limited to,enzymatic fusion proteins, for example, beta-galactosidase fusions,yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-taggedand Ig fusions. Such fusion proteins, particularly poly-His fusions, canfacilitate their purification following recombinant expression. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of a protein can be increased by using a heterologous signalsequence. Fusion proteins are further described in, for example, Terpe,“Overview of tag protein fusions: from molecular and biochemicalfundamentals to commercial systems”, Appl Microbiol Biotechnol. 2003January; 60(5):523-33. Epub 2002 Nov. 7; Graddis et al., “Designingproteins that work using recombinant technologies”, Curr PharmBiotechnol. 2002 December; 3(4):285-97; and Nilsson et al., “Affinityfusion strategies for detection, purification, and immobilization ofrecombinant proteins”, Protein Expr Purif. 1997 October; 11(1): 1-16.

The present invention also relates to further obvious variants of thevariant polypeptides of the present invention, such asnaturally-occurring mature forms (e.g., alleleic variants),non-naturally occurring recombinantly-derived variants, and orthologsand paralogs of such proteins that share sequence homology. Suchvariants can readily be generated using art-known techniques in thefields of recombinant nucleic acid technology and protein biochemistry.It is understood, however, that variants exclude those known in theprior art before the present invention.

Further variants of the variant polypeptides disclosed in Table 1 cancomprise an amino acid sequence that shares at least 70-80%, 80-85%,85-90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identitywith an amino acid sequence disclosed in Table 1 (or a fragment thereof)and that includes a novel amino acid residue (allele) disclosed in Table1 (which is encoded by a novel SNP allele). Thus, an aspect of thepresent invention that is specifically contemplated are polypeptidesthat have a certain degree of sequence variation compared with thepolypeptide sequences shown in Table 1, but that contain a novel aminoacid residue (allele) encoded by a novel SNP allele disclosed herein. Inother words, as long as a polypeptide contains a novel amino acidresidue disclosed herein, other portions of the polypeptide that flankthe novel amino acid residue can vary to some degree from thepolypeptide sequences shown in Table 1.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the amino acid sequences disclosed hereincan readily be identified as having complete sequence identity to one ofthe variant proteins of the present invention as well as being encodedby the same genetic locus as the variant proteins provided herein.

Orthologs of a variant peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof a variant peptide as well as being encoded by a gene from anotherorganism. Preferred orthologs will be isolated from non-human mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs can be encoded by a nucleic acid sequencethat hybridizes to a variant peptide-encoding nucleic acid moleculeunder moderate to stringent conditions depending on the degree ofrelatedness of the two organisms yielding the homologous proteins.

Variant proteins include, but are not limited to, proteins containingdeletions, additions and substitutions in the amino acid sequence causedby the SNPs of the present invention. One class of substitutions isconserved amino acid substitutions in which a given amino acid in apolypeptide is substituted for another amino acid of likecharacteristics. Typical conservative substitutions are replacements,one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in, forexample, Bowie et al., Science 247:1306-1310 (1990).

Variant proteins can be fully functional or can lack function in one ormore activities, e.g. ability to bind another molecule, ability tocatalyze a substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variations orvariations in non-critical residues or in non-critical regions.Functional variants can also contain substitution of similar amino acidsthat result in no change or an insignificant change in function.Alternatively, such substitutions may positively or negatively affectfunction to some degree. Non-functional variants typically contain oneor more non-conservative amino acid substitutions, deletions,insertions, inversions, truncations or extensions, or a substitution,insertion, inversion, or deletion of a critical residue or in a criticalregion.

Amino acids that are essential for function of a protein can beidentified by methods known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science244:1081-1085 (1989)), particularly using the amino acid sequence andpolymorphism information provided in Table 1. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as enzyme activity or in assays such as an in vitro proliferativeactivity. Sites that are critical for binding partner/substrate bindingcan also be determined by structural analysis such as crystallization,nuclear magnetic resonance or photoaffinity labeling (Smith et al., J.Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312(1992)).

Polypeptides can contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Accordingly, the variant proteins of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature polypeptide is fused with anothercompound, such as a compound to increase the half-life of thepolypeptide (e.g., polyethylene glycol), or in which additional aminoacids are fused to the mature polypeptide, such as a leader or secretorysequence or a sequence for purification of the mature polypeptide or apro-protein sequence.

Known protein modifications include, but are not limited to,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such protein modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993); Wold,F., Posttranslational Covalent Modification of Proteins, B. C. Johnson,Ed., Academic Press, New York 1-12 (1983); Seifter et al., Meth.Enzymol. 182: 626-646 (1990); and Rattan et al., Ann. N.Y. Acad. Sci.663:48-62 (1992).

The present invention further provides fragments of the variant proteinsin which the fragments contain one or more amino acid sequencevariations (e.g., substitutions, or truncations or extensions due tocreation or destruction of a stop codon) encoded by one or more SNPsdisclosed herein. The fragments to which the invention pertains,however, are not to be construed as encompassing fragments that havebeen disclosed in the prior art before the present invention.

As used herein, a fragment may comprise at least about 4, 8, 10, 12, 14,16, 18, 20, 25, 30, 50, 100 (or any other number in-between) or morecontiguous amino acid residues from a variant protein, wherein at leastone amino acid residue is affected by a SNP of the present invention,e.g., a variant amino acid residue encoded by a nonsynonymous nucleotidesubstitution at a cSNP position provided by the present invention. Thevariant amino acid encoded by a cSNP may occupy any residue positionalong the sequence of the fragment. Such fragments can be chosen basedon the ability to retain one or more of the biological activities of thevariant protein or the ability to perform a function, e.g., act as animmunogen. Particularly important fragments are biologically activefragments. Such fragments will typically comprise a domain or motif of avariant protein of the present invention, e.g., active site,transmembrane domain, or ligand/substrate binding domain. Otherfragments include, but are not limited to, domain or motif-containingfragments, soluble peptide fragments, and fragments containingimmunogenic structures. Predicted domains and functional sites arereadily identifiable by computer programs well known to those of skillin the art (e.g., PROSITE analysis) (Current Protocols in ProteinScience, John Wiley & Sons, N.Y. (2002)).

Uses of Variant Proteins

The variant proteins of the present invention can be used in a varietyof ways, including but not limited to, in assays to determine thebiological activity of a variant protein, such as in a panel of multipleproteins for high-throughput screening; to raise antibodies or to elicitanother type of immune response; as a reagent (including the labeledreagent) in assays designed to quantitatively determine levels of thevariant protein (or its binding partner) in biological fluids; as amarker for cells or tissues in which it is preferentially expressed(either constitutively or at a particular stage of tissuedifferentiation or development or in a disease state); as a target forscreening for a therapeutic agent; and as a direct therapeutic agent tobe administered into a human subject. Any of the variant proteinsdisclosed herein may be developed into reagent grade or kit format forcommercialization as research products. Methods for performing the useslisted above are well known to those skilled in the art (see, e.g.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Sambrook and Russell, 2000, and Methods in Enzymology: Guide toMolecular Cloning Techniques, Academic Press, Berger, S. L. and A. R.Kimmel eds., 1987).

In a specific embodiment of the invention, the methods of the presentinvention include detection of one or more variant proteins disclosedherein. Variant proteins are disclosed in Table 1 and in the SequenceListing as SEQ ID NOS: 68-134. Detection of such proteins can beaccomplished using, for example, antibodies, small molecule compounds,aptamers, ligands/substrates, other proteins or protein fragments, orother protein-binding agents. Preferably, protein detection agents arespecific for a variant protein of the present invention and cantherefore discriminate between a variant protein of the presentinvention and the wild-type protein or another variant form. This cangenerally be accomplished by, for example, selecting or designingdetection agents that bind to the region of a protein that differsbetween the variant and wild-type protein, such as a region of a proteinthat contains one or more amino acid substitutions that is/are encodedby a non-synonymous cSNP of the present invention, or a region of aprotein that follows a nonsense mutation-type SNP that creates a stopcodon thereby leading to a shorter polypeptide, or a region of a proteinthat follows a read-through mutation-type SNP that destroys a stop codonthereby leading to a longer polypeptide in which a portion of thepolypeptide is present in one version of the polypeptide but not theother.

In another specific aspect of the invention, the variant proteins of thepresent invention are used as targets for diagnosing stenosis or MI orfor determining predisposition to stenosis or MI in a human.Accordingly, the invention provides methods for detecting the presenceof, or levels of, one or more variant proteins of the present inventionin a cell, tissue, or organism. Such methods typically involvecontacting a test sample with an agent (e.g., an antibody, smallmolecule compound, or peptide) capable of interacting with the variantprotein such that specific binding of the agent to the variant proteincan be detected. Such an assay can be provided in a single detectionformat or a multi-detection format such as an array, for example, anantibody or aptamer array (arrays for protein detection may also bereferred to as “protein chips”). The variant protein of interest can beisolated from a test sample and assayed for the presence of a variantamino acid sequence encoded by one or more SNPs disclosed by the presentinvention. The SNPs may cause changes to the protein and thecorresponding protein function/activity, such as through non-synonymoussubstitutions in protein coding regions that can lead to amino acidsubstitutions, deletions, insertions, and/or rearrangements; formationor destruction of stop codons; or alteration of control elements such aspromoters. SNPs may also cause inappropriate post-translationalmodifications.

One preferred agent for detecting a variant protein in a sample is anantibody capable of selectively binding to a variant form of the protein(antibodies are described in greater detail in the next section). Suchsamples include, for example, tissues, cells, and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject.

In vitro methods for detection of the variant proteins associated withMI that are disclosed herein and fragments thereof include, but are notlimited to, enzyme linked immunosorbent assays (ELISAs),radioimmunoassays (RIA), Western blots, immunoprecipitations,immunofluorescence, and protein arrays/chips (e.g., arrays of antibodiesor aptamers). For further information regarding immunoassays and relatedprotein detection methods, see Current Protocols in Immunology, JohnWiley & Sons, N.Y., and Hage, “Immunoassays”, Anal Chem. 1999 Jun. 15;71(12):294R-304R.

Additional analytic methods of detecting amino acid variants include,but are not limited to, altered electrophoretic mobility, alteredtryptic peptide digest, altered protein activity in cell-based orcell-free assay, alteration in ligand or antibody-binding pattern,altered isoelectric point, and direct amino acid sequencing.

Alternatively, variant proteins can be detected in vivo in a subject byintroducing into the subject a labeled antibody (or other type ofdetection reagent) specific for a variant protein. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

Other uses of the variant peptides of the present invention are based onthe class or action of the protein. For example, proteins isolated fromhumans and their mammalian orthologs serve as targets for identifyingagents (e.g., small molecule drugs or antibodies) for use in therapeuticapplications, particularly for modulating a biological or pathologicalresponse in a cell or tissue that expresses the protein. Pharmaceuticalagents can be developed that modulate protein activity.

As an alternative to modulating gene expression, therapeutic compoundscan be developed that modulate protein function. For example, many SNPsdisclosed herein affect the amino acid sequence of the encoded protein(e.g., non-synonymous cSNPs and nonsense mutation-type SNPs). Suchalterations in the encoded amino acid sequence may affect proteinfunction, particularly if such amino acid sequence variations occur infunctional protein domains, such as catalytic domains, ATP-bindingdomains, or ligand/substrate binding domains. It is well established inthe art that variant proteins having amino acid sequence variations infunctional domains can cause or influence pathological conditions. Insuch instances, compounds (e.g., small molecule drugs or antibodies) canbe developed that target the variant protein and modulate (e.g., up- ordown-regulate) protein function/activity.

The therapeutic methods of the present invention further include methodsthat target one or more variant proteins of the present invention.Variant proteins can be targeted using, for example, small moleculecompounds, antibodies, aptamers, ligands/substrates, other proteins, orother protein-binding agents. Additionally, the skilled artisan willrecognize that the novel protein variants (and polymorphic nucleic acidmolecules) disclosed in Table 1 may themselves be directly used astherapeutic agents by acting as competitive inhibitors of correspondingart-known proteins (or nucleic acid molecules such as mRNA molecules).

The variant proteins of the present invention are particularly useful indrug screening assays, in cell-based or cell-free systems. Cell-basedsystems can utilize cells that naturally express the protein, a biopsyspecimen, or cell cultures. In one embodiment, cell-based assays involverecombinant host cells expressing the variant protein. Cell-free assayscan be used to detect the ability of a compound to directly bind to avariant protein or to the corresponding SNP-containing nucleic acidfragment that encodes the variant protein.

A variant protein of the present invention, as well as appropriatefragments thereof, can be used in high-throughput screening assays totest candidate compounds for the ability to bind and/or modulate theactivity of the variant protein. These candidate compounds can befurther screened against a protein having normal function (e.g., awild-type/non-variant protein) to further determine the effect of thecompound on the protein activity. Furthermore, these compounds can betested in animal or invertebrate systems to determine in vivoactivity/effectiveness. Compounds can be identified that activate(agonists) or inactivate (antagonists) the variant protein, anddifferent compounds can be identified that cause various degrees ofactivation or inactivation of the variant protein.

Further, the variant proteins can be used to screen a compound for theability to stimulate or inhibit interaction between the variant proteinand a target molecule that normally interacts with the protein. Thetarget can be a ligand, a substrate or a binding partner that theprotein normally interacts with (for example, epinephrine ornorepinephrine). Such assays typically include the steps of combiningthe variant protein with a candidate compound under conditions thatallow the variant protein, or fragment thereof, to interact with thetarget molecule, and to detect the formation of a complex between theprotein and the target or to detect the biochemical consequence of theinteraction with the variant protein and the target, such as any of theassociated effects of signal transduction.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the variant protein thatcompetes for ligand binding. Other candidate compounds include mutantproteins or appropriate fragments containing mutations that affectvariant protein function and thus compete for ligand. Accordingly, afragment that competes for ligand, for example with a higher affinity,or a fragment that binds ligand but does not allow release, isencompassed by the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) variant protein activity.The assays typically involve an assay of events in the signaltransduction pathway that indicate protein activity. Thus, theexpression of genes that are up or down-regulated in response to thevariant protein dependent signal cascade can be assayed. In oneembodiment, the regulatory region of such genes can be operably linkedto a marker that is easily detectable, such as luciferase.Alternatively, phosphorylation of the variant protein, or a variantprotein target, could also be measured. Any of the biological orbiochemical functions mediated by the variant protein can be used as anendpoint assay. These include all of the biochemical or biologicalevents described herein, in the references cited herein, incorporated byreference for these endpoint assay targets, and other functions known tothose of ordinary skill in the art.

Binding and/or activating compounds can also be screened by usingchimeric variant proteins in which an amino terminal extracellulardomain or parts thereof, an entire transmembrane domain or subregions,and/or the carboxyl terminal intracellular domain or parts thereof, canbe replaced by heterologous domains or subregions. For example, asubstrate-binding region can be used that interacts with a differentsubstrate than that which is normally recognized by a variant protein.Accordingly, a different set of signal transduction components isavailable as an end-point assay for activation. This allows for assaysto be performed in other than the specific host cell from which thevariant protein is derived.

The variant proteins are also useful in competition binding assays inmethods designed to discover compounds that interact with the variantprotein. Thus, a compound can be exposed to a variant protein underconditions that allow the compound to bind or to otherwise interact withthe variant protein. A binding partner, such as ligand, that normallyinteracts with the variant protein is also added to the mixture. If thetest compound interacts with the variant protein or its binding partner,it decreases the amount of complex formed or activity from the variantprotein. This type of assay is particularly useful in screening forcompounds that interact with specific regions of the variant protein(Hodgson, Bio/technology, 1992, Sep. 10(9), 973-80).

To perform cell-free drug screening assays, it is sometimes desirable toimmobilize either the variant protein or a fragment thereof, or itstarget molecule, to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Any method for immobilizing proteins onmatrices can be used in drug screening assays. In one embodiment, afusion protein containing an added domain allows the protein to be boundto a matrix. For example, glutathione-S-transferase/¹²⁵I fusion proteinscan be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.Louis, Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and a candidatecompound, such as a drug candidate, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads can bewashed to remove any unbound label, and the matrix immobilized andradiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofbound material found in the bead fraction quantitated from the gel usingstandard electrophoretic techniques.

Either the variant protein or its target molecule can be immobilizedutilizing conjugation of biotin and streptavidin. Alternatively,antibodies reactive with the variant protein but which do not interferewith binding of the variant protein to its target molecule can bederivatized to the wells of the plate, and the variant protein trappedin the wells by antibody conjugation. Preparations of the targetmolecule and a candidate compound are incubated in the variantprotein-presenting wells and the amount of complex trapped in the wellcan be quantitated. Methods for detecting such complexes, in addition tothose described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the proteintarget molecule, or which are reactive with variant protein and competewith the target molecule, and enzyme-linked assays that rely ondetecting an enzymatic activity associated with the target molecule.

Modulators of variant protein activity identified according to thesedrug screening assays can be used to treat a subject with a disordermediated by the protein pathway, such as MI. These methods of treatmenttypically include the steps of administering the modulators of proteinactivity in a pharmaceutical composition to a subject in need of suchtreatment.

The variant proteins, or fragments thereof, disclosed herein canthemselves be directly used to treat a disorder characterized by anabsence of, inappropriate, or unwanted expression or activity of thevariant protein. Accordingly, methods for treatment include the use of avariant protein disclosed herein or fragments thereof.

In yet another aspect of the invention, variant proteins can be used as“bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300) to identify other proteins that bind to orinteract with the variant protein and are involved in variant proteinactivity. Such variant protein-binding proteins are also likely to beinvolved in the propagation of signals by the variant proteins orvariant protein targets as, for example, elements of a protein-mediatedsignaling pathway. Alternatively, such variant protein-binding proteinsare inhibitors of the variant protein.

The two-hybrid system is based on the modular nature of mosttranscription factors, which typically consist of separable DNA-bindingand activation domains. Briefly, the assay typically utilizes twodifferent DNA constructs. In one construct, the gene that codes for avariant protein is fused to a gene encoding the DNA binding domain of aknown transcription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming a variantprotein-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) that is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detected,and cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene that encodes the proteinthat interacts with the variant protein.

Antibodies Directed to Variant Proteins

The present invention also provides antibodies that selectively bind tothe variant proteins disclosed herein and fragments thereof. Suchantibodies may be used to quantitatively or qualitatively detect thevariant proteins of the present invention. As used herein, an antibodyselectively binds a target variant protein when it binds the variantprotein and does not significantly bind to non-variant proteins, i.e.,the antibody does not significantly bind to normal, wild-type, orart-known proteins that do not contain a variant amino acid sequence dueto one or more SNPs of the present invention (variant amino acidsequences may be due to, for example, nonsynonymous cSNPs, nonsense SNPsthat create a stop codon, thereby causing a truncation of a polypeptideor SNPs that cause read-through mutations resulting in an extension of apolypeptide).

As used herein, an antibody is defined in terms consistent with thatrecognized in the art: they are multi-subunit proteins produced by anorganism in response to an antigen challenge. The antibodies of thepresent invention include both monoclonal antibodies and polyclonalantibodies, as well as antigen-reactive proteolytic fragments of suchantibodies, such as Fab, F(ab)′₂, and Fv fragments. In addition, anantibody of the present invention further includes any of a variety ofengineered antigen-binding molecules such as a chimeric antibody (U.S.Pat. Nos. 4,816,567 and 4,816,397; Morrison et al., Proc. Natl. Acad.Sci. USA, 81:6851, 1984; Neuberger et al., Nature 312:604, 1984), ahumanized antibody (U.S. Pat. Nos. 5,693,762; 5,585,089; and 5,565,332),a single-chain Fv (U.S. Pat. No. 4,946,778; Ward et al., Nature 334:544,1989), a bispecific antibody with two binding specificities (Segal etal., J. Immunol Methods 248:1, 2001; Carter, J. Immunol. Methods 248:7,2001), a diabody, a triabody, and a tetrabody (Todorovska et al., J.Immunol. Methods, 248:47, 2001), as well as a Fab conjugate (dimer ortrimer), and a minibody.

Many methods are known in the art for generating and/or identifyingantibodies to a given target antigen (Harlow, Antibodies, Cold SpringHarbor Press, (1989)). In general, an isolated peptide (e.g., a variantprotein of the present invention) is used as an immunogen and isadministered to a mammalian organism, such as a rat, rabbit, hamster ormouse. Either a full-length protein, an antigenic peptide fragment(e.g., a peptide fragment containing a region that varies between avariant protein and a corresponding wild-type protein), or a fusionprotein can be used. A protein used as an immunogen may benaturally-occurring, synthetic or recombinantly produced, and may beadministered in combination with an adjuvant, including but not limitedto, Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substance such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and the like.

Monoclonal antibodies can be produced by hybridoma technology (Kohlerand Milstein, Nature, 256:495, 1975), which immortalizes cells secretinga specific monoclonal antibody. The immortalized cell lines can becreated in vitro by fusing two different cell types, typicallylymphocytes, and tumor cells. The hybridoma cells may be cultivated invitro or in vivo. Additionally, fully human antibodies can be generatedby transgenic animals (He et al., J. Immunol., 169:595, 2002). Fd phageand Fd phagemid technologies may be used to generate and selectrecombinant antibodies in vitro (Hoogenboom and Chames, Immunol. Today21:371, 2000; Liu et al., J. Mol. Biol. 315:1063, 2002). Thecomplementarity-determining regions of an antibody can be identified,and synthetic peptides corresponding to such regions may be used tomediate antigen binding (U.S. Pat. No. 5,637,677).

Antibodies are preferably prepared against regions or discrete fragmentsof a variant protein containing a variant amino acid sequence ascompared to the corresponding wild-type protein (e.g., a region of avariant protein that includes an amino acid encoded by a nonsynonymouscSNP, a region affected by truncation caused by a nonsense SNP thatcreates a stop codon, or a region resulting from the destruction of astop codon due to read-through mutation caused by a SNP). Furthermore,preferred regions will include those involved in function/activityand/or protein/binding partner interaction. Such fragments can beselected on a physical property, such as fragments corresponding toregions that are located on the surface of the protein, e.g.,hydrophilic regions, or can be selected based on sequence uniqueness, orbased on the position of the variant amino acid residue(s) encoded bythe SNPs provided by the present invention. An antigenic fragment willtypically comprise at least about 8-10 contiguous amino acid residues inwhich at least one of the amino acid residues is an amino acid affectedby a SNP disclosed herein. The antigenic peptide can comprise, however,at least 12, 14, 16, 20, 25, 50, 100 (or any other number in-between) ormore amino acid residues, provided that at least one amino acid isaffected by a SNP disclosed herein.

Detection of an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody or an antigen-reactivefragment thereof to a detectable substance. Detectable substancesinclude, but are not limited to, various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibodies, particularly the use of antibodies as therapeutic agents,are reviewed in: Morgan, “Antibody therapy for Alzheimer's disease,”Expert Rev Vaccines. 2003 February; 2(1):53-9; Ross et al., “Anticancerantibodies,” Am J Clin Pathol. 2003 April; 119(4):472-85; Goldenberg,“Advancing role of radiolabeled antibodies in the therapy of cancer,”Cancer Immunol Immunother. 2003 May; 52(5):281-96. Epub 2003 Mar. 11;Ross et al., “Antibody-based therapeutics in oncology,” Expert RevAnticancer Ther. 2003 February; 3(1):107-21; Cao et al., “Bispecificantibody conjugates in therapeutics,” Adv Drug Deliv Rev. 2003 Feb. 10;55(2):171-97; von Mehren et al., “Monoclonal antibody therapy forcancer,” Annu Rev Med. 2003; 54:343-69. Epub 2001 Dec. 3; Hudson et al.,“Engineered antibodies,” Nat Med. 2003 January; 9(1):129-34; Brekke etal., “Therapeutic antibodies for human diseases at the dawn of thetwenty-first century,” Nat Rev Drug Discov. 2003 January; 2(1):52-62(Erratum in: Nat Rev Drug Discov. 2003 March; 2(3):240); Houdebine,“Antibody manufacture in transgenic animals and comparisons with othersystems,” Curr Opin Biotechnol. 2002 December; 113(6):625-9; Andreakoset al., “Monoclonal antibodies in immune and inflammatory diseases,”Curr Opin Biotechnol. 2002 December; 13(6):615-20; Kellermann et al.,“Antibody discovery: the use of transgenic mice to generate humanmonoclonal antibodies for therapeutics,” Curr Opin Biotechnol. 2002December; 13(6):593-7; Pini et al., “Phage display and colony filterscreening for high-throughput selection of antibody libraries,” CombChem High Throughput Screen. 2002 November; 5(7):503-10; Batra et al.,“Pharmacokinetics and biodistribution of genetically engineeredantibodies,” Curr Opin Biotechnol. 2002 December; 13(6):603-8; andTangri et al., “Rationally engineered proteins or antibodies with absentor reduced immunogenicity,” Curr Med. Chem. 2002 December; 9(24):2191-9.

Uses of Antibodies

Antibodies can be used to isolate the variant proteins of the presentinvention from a natural cell source or from recombinant host cells bystandard techniques, such as affinity chromatography orimmunoprecipitation. In addition, antibodies are useful for detectingthe presence of a variant protein of the present invention in cells ortissues to determine the pattern of expression of the variant proteinamong various tissues in an organism and over the course of normaldevelopment or disease progression. Further, antibodies can be used todetect variant protein in situ, in vitro, in a bodily fluid, or in acell lysate or supernatant in order to evaluate the amount and patternof expression. Also, antibodies can be used to assess abnormal tissuedistribution, abnormal expression during development, or expression inan abnormal condition, such as MI. Additionally, antibody detection ofcirculating fragments of the full-length variant protein can be used toidentify turnover.

Antibodies to the variant proteins of the present invention are alsouseful in pharmacogenomic analysis. Thus, antibodies against variantproteins encoded by alternative SNP alleles can be used to identifyindividuals that require modified treatment modalities.

Further, antibodies can be used to assess expression of the variantprotein in disease states such as in active stages of the disease or inan individual with a predisposition to a disease related to theprotein's function, particularly stenosis or MI. Antibodies specific fora variant protein encoded by a SNP-containing nucleic acid molecule ofthe present invention can be used to assay for the presence of thevariant protein, such as to screen for predisposition to MI as indicatedby the presence of the variant protein.

Antibodies are also useful as diagnostic tools for evaluating thevariant proteins in conjunction with analysis by electrophoreticmobility, isoelectric point, tryptic peptide digest, and other physicalassays well known in the art.

Antibodies are also useful for tissue typing. Thus, where a specificvariant protein has been correlated with expression in a specifictissue, antibodies that are specific for this protein can be used toidentify a tissue type.

Antibodies can also be used to assess aberrant subcellular localizationof a variant protein in cells in various tissues. The diagnostic usescan be applied, not only in genetic testing, but also in monitoring atreatment modality. Accordingly, where treatment is ultimately aimed atcorrecting the expression level or the presence of variant protein oraberrant tissue distribution or developmental expression of a variantprotein, antibodies directed against the variant protein or relevantfragments can be used to monitor therapeutic efficacy.

The antibodies are also useful for inhibiting variant protein function,for example, by blocking the binding of a variant protein to a bindingpartner. These uses can also be applied in a therapeutic context inwhich treatment involves inhibiting a variant protein's function. Anantibody can be used, for example, to block or competitively inhibitbinding, thus modulating (agonizing or antagonizing) the activity of avariant protein. Antibodies can be prepared against specific variantprotein fragments containing sites required for function or against anintact variant protein that is associated with a cell or cell membrane.For in vivo administration, an antibody may be linked with an additionaltherapeutic payload such as a radionuclide, an enzyme, an immunogenicepitope, or a cytotoxic agent. Suitable cytotoxic agents include, butare not limited to, bacterial toxin such as diphtheria, and plant toxinsuch as ricin. The in vivo half-life of an antibody or a fragmentthereof may be lengthened by pegylation through conjugation topolyethylene glycol (Leong et al., Cytokine 16:106, 2001).

The invention also encompasses kits for using antibodies, such as kitsfor detecting the presence of a variant protein in a test sample. Anexemplary kit can comprise antibodies such as a labeled or labelableantibody and a compound or agent for detecting variant proteins in abiological sample; means for determining the amount, or presence/absenceof variant protein in the sample; means for comparing the amount ofvariant protein in the sample with a standard; and instructions for use.

Vectors and Host Cells

The present invention also provides vectors containing theSNP-containing nucleic acid molecules described herein. The term“vector” refers to a vehicle, preferably a nucleic acid molecule, whichcan transport a SNP-containing nucleic acid molecule. When the vector isa nucleic acid molecule, the SNP-containing nucleic acid molecule can becovalently linked to the vector nucleic acid. Such vectors include, butare not limited to, a plasmid, single or double stranded phage, a singleor double stranded RNA or DNA viral vector, or artificial chromosome,such as a BAC, PAC, YAC, or MAC.

A vector can be maintained in a host cell as an extrachromosomal elementwhere it replicates and produces additional copies of the SNP-containingnucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the SNP-containingnucleic acid molecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the SNP-containingnucleic acid molecules. The vectors can function in prokaryotic oreukaryotic cells or in both (shuttle vectors).

Expression vectors typically contain cis-acting regulatory regions thatare operably linked in the vector to the SNP-containing nucleic acidmolecules such that transcription of the SNP-containing nucleic acidmolecules is allowed in a host cell. The SNP-containing nucleic acidmolecules can also be introduced into the host cell with a separatenucleic acid molecule capable of affecting transcription. Thus, thesecond nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the SNP-containing nucleic acid molecules from thevector. Alternatively, a trans-acting factor may be supplied by the hostcell. Finally, a trans-acting factor can be produced from the vectoritself. It is understood, however, that in some embodiments,transcription and/or translation of the nucleic acid molecules can occurin a cell-free system.

The regulatory sequences to which the SNP-containing nucleic acidmolecules described herein can be operably linked include promoters fordirecting mRNA transcription. These include, but are not limited to, theleft promoter from bacteriophage λ, the lac, TRP, and TAC promoters fromE. coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region, aribosome-binding site for translation. Other regulatory control elementsfor expression include initiation and termination codons as well aspolyadenylation signals. A person of ordinary skill in the art would beaware of the numerous regulatory sequences that are useful in expressionvectors (see, e.g., Sambrook and Russell, 2000, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

A variety of expression vectors can be used to express a SNP-containingnucleic acid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example, vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors can also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g., cosmids and phagemids. Appropriate cloning andexpression vectors for prokaryotic and eukaryotic hosts are described inSambrook and Russell, 2000, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The regulatory sequence in a vector may provide constitutive expressionin one or more host cells (e.g., tissue specific expression) or mayprovide for inducible expression in one or more cell types such as bytemperature, nutrient additive, or exogenous factor, e.g., a hormone orother ligand. A variety of vectors that provide constitutive orinducible expression of a nucleic acid sequence in prokaryotic andeukaryotic host cells are well known to those of ordinary skill in theart.

A SNP-containing nucleic acid molecule can be inserted into the vectorby methodology well-known in the art. Generally, the SNP-containingnucleic acid molecule that will ultimately be expressed is joined to anexpression vector by cleaving the SNP-containing nucleic acid moleculeand the expression vector with one or more restriction enzymes and thenligating the fragments together. Procedures for restriction enzymedigestion and ligation are well known to those of ordinary skill in theart.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial host cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic host cells include, but are not limited to, yeast, insectcells such as Drosophila, animal cells such as COS and CHO cells, andplant cells.

As described herein, it may be desirable to express the variant peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the variant peptides. Fusion vectorscan, for example, increase the expression of a recombinant protein,increase the solubility of the recombinant protein, and aid in thepurification of the protein by acting, for example, as a ligand foraffinity purification. A proteolytic cleavage site may be introduced atthe junction of the fusion moiety so that the desired variant peptidecan ultimately be separated from the fusion moiety. Proteolytic enzymessuitable for such use include, but are not limited to, factor Xa,thrombin, and enterokinase. Typical fusion expression vectors includepGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in a bacterial host byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe SNP-containing nucleic acid molecule of interest can be altered toprovide preferential codon usage for a specific host cell, for example,E. coli (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The SNP-containing nucleic acid molecules can also be expressed byexpression vectors that are operative in yeast. Examples of vectors forexpression in yeast (e.g., S. cerevisiae) include pYepSec1 (Baldari, etal., EMBO J. 6:229-234 (1987)), pMFa (Kujan et al., Cell30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), andpYES2 (Invitrogen Corporation, San Diego, Calif.).

The SNP-containing nucleic acid molecules can also be expressed ininsect cells using, for example, baculovirus expression vectors.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.,Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al.,Virology 170:31-39 (1989)).

In certain embodiments of the invention, the SNP-containing nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

The invention also encompasses vectors in which the SNP-containingnucleic acid molecules described herein are cloned into the vector inreverse orientation, but operably linked to a regulatory sequence thatpermits transcription of antisense RNA. Thus, an antisense transcriptcan be produced to the SNP-containing nucleic acid sequences describedherein, including both coding and non-coding regions. Expression of thisantisense RNA is subject to each of the parameters described above inrelation to expression of the sense RNA (regulatory sequences,constitutive or inducible expression, tissue-specific expression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include, for example,prokaryotic cells, lower eukaryotic cells such as yeast, othereukaryotic cells such as insect cells, and higher eukaryotic cells suchas mammalian cells.

The recombinant host cells can be prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to persons of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those described in Sambrook and Russell, 2000,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Host cells can contain more than one vector. Thus, differentSNP-containing nucleotide sequences can be introduced in differentvectors into the same cell. Similarly, the SNP-containing nucleic acidmolecules can be introduced either alone or with other nucleic acidmolecules that are not related to the SNP-containing nucleic acidmolecules, such as those providing trans-acting factors for expressionvectors. When more than one vector is introduced into a cell, thevectors can be introduced independently, co-introduced, or joined to thenucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication can occur in host cells that provide functionsthat complement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be inserted in the same vector that containsthe SNP-containing nucleic acid molecules described herein or may be ina separate vector. Markers include, for example, tetracycline orampicillin-resistance genes for prokaryotic host cells, anddihydrofolate reductase or neomycin resistance genes for eukaryotic hostcells. However, any marker that provides selection for a phenotypictrait can be effective.

While the mature variant proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these variant proteins using RNA derivedfrom the DNA constructs described herein.

Where secretion of the variant protein is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such asG-protein-coupled receptors (GPCRs), appropriate secretion signals canbe incorporated into the vector. The signal sequence can be endogenousto the peptides or heterologous to these peptides.

Where the variant protein is not secreted into the medium, the proteincan be isolated from the host cell by standard disruption procedures,including freeze/thaw, sonication, mechanical disruption, use of lysingagents, and the like. The variant protein can then be recovered andpurified by well-known purification methods including, for example,ammonium sulfate precipitation, acid extraction, anion or cationicexchange chromatography, phosphocellulose chromatography,hydrophobic-interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography.

It is also understood that, depending upon the host cell in whichrecombinant production of the variant proteins described herein occurs,they can have various glycosylation patterns, or may benon-glycosylated, as when produced in bacteria. In addition, the variantproteins may include an initial modified methionine in some cases as aresult of a host-mediated process.

For further information regarding vectors and host cells, see CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y.

Uses of Vectors and Host Cells, and Transgenic Animals

Recombinant host cells that express the variant proteins describedherein have a variety of uses. For example, the cells are useful forproducing a variant protein that can be further purified into apreparation of desired amounts of the variant protein or fragmentsthereof. Thus, host cells containing expression vectors are useful forvariant protein production.

Host cells are also useful for conducting cell-based assays involvingthe variant protein or variant protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a variant protein is useful forassaying compounds that stimulate or inhibit variant protein function.Such an ability of a compound to modulate variant protein function maynot be apparent from assays of the compound on the native/wild-typeprotein, or from cell-free assays of the compound. Recombinant hostcells are also useful for assaying functional alterations in the variantproteins as compared with a known function.

Genetically-engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably anon-human mammal, for example, a rodent, such as a rat or mouse, inwhich one or more of the cells of the animal include a transgene. Atransgene is exogenous DNA containing a SNP of the present inventionwhich is integrated into the genome of a cell from which a transgenicanimal develops and which remains in the genome of the mature animal inone or more of its cell types or tissues. Such animals are useful forstudying the function of a variant protein in vivo, and identifying andevaluating modulators of variant protein activity. Other examples oftransgenic animals include, but are not limited to, non-human primates,sheep, dogs, cows, goats, chickens, and amphibians. Transgenic non-humanmammals such as cows and goats can be used to produce variant proteinswhich can be secreted in the animal's milk and then recovered.

A transgenic animal can be produced by introducing a SNP-containingnucleic acid molecule into the male pronuclei of a fertilized oocyte,e.g., by microinjection or retroviral infection, and allowing the oocyteto develop in a pseudopregnant female foster animal. Any nucleic acidmolecules that contain one or more SNPs of the present invention canpotentially be introduced as a transgene into the genome of a non-humananimal.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the variant protein in particularcells or tissues.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described in, for example, U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al., and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes a non-human animal in which the entire animal or tissuesin the animal have been produced using the homologously recombinant hostcells described herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1 (Lakso et al. PNAS 89:6232-6236 (1992)).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991)). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein are generally needed. Such animalscan be provided through the construction of “double” transgenic animals,e.g., by mating two transgenic animals, one containing a transgeneencoding a selected variant protein and the other containing a transgeneencoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in, for example, Wilmut, I.et al. Nature 385:810-813 (1997) and PCT International Publication Nos.WO 97/07668 and WO 97/07669. In brief, a cell (e.g., a somatic cell)from the transgenic animal can be isolated and induced to exit thegrowth cycle and enter G_(o) phase. The quiescent cell can then befused, e.g., through the use of electrical pulses, to an enucleatedoocyte from an animal of the same species from which the quiescent cellis isolated. The reconstructed oocyte is then cultured such that itdevelops to morula or blastocyst and then transferred to pseudopregnantfemale foster animal. The offspring born of this female foster animalwill be a clone of the animal from which the cell (e.g., a somatic cell)is isolated.

Transgenic animals containing recombinant cells that express the variantproteins described herein are useful for conducting the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could influence ligand orsubstrate binding, variant protein activation, signal transduction, orother processes or interactions, may not be evident from in vitrocell-free or cell-based assays. Thus, non-human transgenic animals ofthe present invention may be used to assay in vivo variant proteinfunction as well as the activities of a therapeutic agent or compoundthat modulates variant protein function/activity or expression. Suchanimals are also suitable for assessing the effects of null mutations(i.e., mutations that substantially or completely eliminate one or morevariant protein functions).

For further information regarding transgenic animals, see Houdebine,“Antibody manufacture in transgenic animals and comparisons with othersystems,” Curr Opin Biotechnol. 2002 December; 13(6):625-9; Petters etal., “Transgenic animals as models for human disease,” Transgenic Res.2000; 9(4-5):347-51; discussion 345-6; Wolf et al., “Use of transgenicanimals in understanding molecular mechanisms of toxicity,” J PharmPharmacol. 1998 June; 50(6):567-74; Echelard, “Recombinant proteinproduction in transgenic animals,” Curr Opin Biotechnol. 1996 October;7(5):536-40; Houdebine, “Transgenic animal bioreactors,” Transgenic Res.2000; 9(4-5):305-20; Pirity et al., “Embryonic stem cells, creatingtransgenic animals,” Methods Cell Biol. 1998; 57:279-93; and Robl etal., “Artificial chromosome vectors and expression of complex proteinsin transgenic animals,” Theriogenology. 2003 Jan. 1; 59(1):107-13.

Computer-Related Embodiments

The SNPs provided in the present invention may be “provided” in avariety of mediums to facilitate use thereof. As used in this section,“provided” refers to a manufacture, other than an isolated nucleic acidmolecule, that contains SNP information of the present invention. Such amanufacture provides the SNP information in a form that allows a skilledartisan to examine the manufacture using means not directly applicableto examining the SNPs or a subset thereof as they exist in nature or inpurified form. The SNP information that may be provided in such a formincludes any of the SNP information provided by the present inventionsuch as, for example, polymorphic nucleic acid and/or amino acidsequence information such as SEQ ID NOS: 1-67, SEQ ID NOS: 68-134, SEQID NOS: 229-299, SEQ ID NOS: 135-228, and SEQ ID NOS: 300-504;information about observed SNP alleles, alternative codons, populations,allele frequencies, SNP types, and/or affected proteins; or any otherinformation provided by the present invention in Tables 1-2 and/or theSequence Listing.

In one application of this embodiment, the SNPs of the present inventioncan be recorded on a computer readable medium. As used herein, “computerreadable medium” refers to any medium that can be read and accesseddirectly by a computer. Such media include, but are not limited to:magnetic storage media, such as floppy discs, hard disc storage medium,and magnetic tape; optical storage media such as CD-ROM; electricalstorage media such as RAM and ROM; and hybrids of these categories suchas magnetic/optical storage media. A skilled artisan can readilyappreciate how any of the presently known computer readable media can beused to create a manufacture comprising computer readable medium havingrecorded thereon a nucleotide sequence of the present invention. Onesuch medium is provided with the present application, namely, thepresent application contains computer readable medium (CD-R) that hasnucleic acid sequences (and encoded protein sequences) containing SNPsprovided/recorded thereon in ASCII text format in a Sequence Listingalong with accompanying Tables that contain detailed SNP and relatedsequence information (transcript sequences are referred to as SEQ IDNOS: 1-67, protein sequences are provided as SEQ ID NOS: 68-134, genomicsequences are provided as SEQ ID NOS: 229-299, transcript-based contextsequences are provided as SEQ ID NOS: 135-228, and genomic-based contextsequences are provided as SEQ ID NOS: 300-504).

As used herein, “recorded” refers to a process for storing informationon computer readable medium. A skilled artisan can readily adopt any ofthe presently known methods for recording information on computerreadable medium to generate manufactures comprising the SNP informationof the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotide/aminoacid sequence information of the present invention on computer readablemedium. For example, the sequence information can be represented in aword processing text file, formatted in commercially-available softwaresuch as WordPerfect and Microsoft Word, represented in the form of anASCII file, or stored in a database application, such as OB2, Sybase,Oracle, or the like. A skilled artisan can readily adapt any number ofdata processor structuring formats (e.g., text file or database) inorder to obtain computer readable medium having recorded thereon the SNPinformation of the present invention.

By providing the SNPs of the present invention in computer readableform, a skilled artisan can routinely access the SNP information for avariety of purposes. Computer software is publicly available whichallows a skilled artisan to access sequence information provided in acomputer readable medium. Examples of publicly available computersoftware include BLAST (Altschul et al., J. Mol. Biol. 215:403-410(1990)) and BLAZE (Brutlag et al., Comp. Chem. 17:203-207 (1993)) searchalgorithms.

The present invention further provides systems, particularlycomputer-based systems, which contain the SNP information describedherein. Such systems may be designed to store and/or analyze informationon, for example, a large number of SNP positions, or information on SNPgenotypes from a large number of individuals. The SNP information of thepresent invention represents a valuable information source. The SNPinformation of the present invention stored/analyzed in a computer-basedsystem may be used for such computer-intensive applications asdetermining or analyzing SNP allele frequencies in a population, mappingdisease genes, genotype-phenotype association studies, grouping SNPsinto haplotypes, correlating SNP haplotypes with response to particulardrugs, or for various other bioinformatic, pharmacogenomic, drugdevelopment, or human identification/forensic applications.

As used herein, “a computer-based system” refers to the hardware means,software means, and data storage means used to analyze the SNPinformation of the present invention. The minimum hardware means of thecomputer-based systems of the present invention typically comprises acentral processing unit (CPU), input means, output means, and datastorage means. A skilled artisan can readily appreciate that any one ofthe currently available computer-based systems are suitable for use inthe present invention. Such a system can be changed into a system of thepresent invention by utilizing the SNP information provided on the CD-R,or a subset thereof, without any experimentation.

As stated above, the computer-based systems of the present inventioncomprise a data storage means having stored therein SNPs of the presentinvention and the necessary hardware means and software means forsupporting and implementing a search means. As used herein, “datastorage means” refers to memory which can store SNP information of thepresent invention, or a memory access means which can accessmanufactures having recorded thereon the SNP information of the presentinvention.

As used herein, “search means” refers to one or more programs oralgorithms that are implemented on the computer-based system to identifyor analyze SNPs in a target sequence based on the SNP information storedwithin the data storage means. Search means can be used to determinewhich nucleotide is present at a particular SNP position in the targetsequence. As used herein, a “target sequence” can be any DNA sequencecontaining the SNP position(s) to be searched or queried.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequencescontaining a SNP position in which the sequence(s) is chosen based on athree-dimensional configuration that is formed upon the folding of thetarget motif. There are a variety of target motifs known in the art.Protein target motifs include, but are not limited to, enzymatic activesites and signal sequences. Nucleic acid target motifs include, but arenot limited to, promoter sequences, hairpin structures, and inducibleexpression elements (protein binding sequences).

A variety of structural formats for the input and output means can beused to input and output the information in the computer-based systemsof the present invention. An exemplary format for an output means is adisplay that depicts the presence or absence of specified nucleotides(alleles) at particular SNP positions of interest. Such presentation canprovide a rapid, binary scoring system for many SNPs simultaneously.

One exemplary embodiment of a computer-based system comprising SNPinformation of the present invention is provided in FIG. 1. FIG. 1provides a block diagram of a computer system 102 that can be used toimplement the present invention. The computer system 102 includes aprocessor 106 connected to a bus 104. Also connected to the bus 104 area main memory 108 (preferably implemented as random access memory, RAM)and a variety of secondary storage devices 110, such as a hard drive 112and a removable medium storage device 114. The removable medium storagedevice 114 may represent, for example, a floppy disk drive, a CD-ROMdrive, a magnetic tape drive, etc. A removable storage medium 116 (suchas a floppy disk, a compact disk, a magnetic tape, etc.) containingcontrol logic and/or data recorded therein may be inserted into theremovable medium storage device 114. The computer system 102 includesappropriate software for reading the control logic and/or the data fromthe removable storage medium 116 once inserted in the removable mediumstorage device 114.

The SNP information of the present invention may be stored in awell-known manner in the main memory 108, any of the secondary storagedevices 110, and/or a removable storage medium 116. Software foraccessing and processing the SNP information (such as SNP scoring tools,search tools, comparing tools, etc.) preferably resides in main memory108 during execution.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1 Statistical Analysis of SNP Allelic and Genotypic Associationwith MI Risk

A case-control genetic study was performed to determine the associationof SNPs in the human genome with coronary heart disease (CHD), and inparticular myocardial infarction (MI), using genomic DNA extracted fromtwo independently obtained sample sets. One set was obtained from theUniversity of California at San Francisco (UCSF). Only individualsself-described as Caucasian were used, in order to minimize thelikelihood of population stratification. Individuals included patientswho had undergone diagnostic or interventional cardiac catheterization,patients at the UCSF Lipid Clinic, and healthy older individuals. Thesamples were divided into cases and controls based on study design(Table 4). Cases were individuals with a history of MI, and controlswere individuals with no symptomatic cardiovascular disease (CVD). CVDin this study refers to such conditions as unstable angina, coronaryaneurysm, CHD such as MI, etc. MI was defined as a medical diagnosis ofICD9 code 410 or 411 (World Heath Organization's InternationalClassification of Disease, 9th Revision), by a clinical chart review orself-reported history. Efforts by UCSF physicians to clinically verifyself-reported MI have resulted in better than 98% verification. For eachparticular SNP association study, a subset of the case and controlpopulations were investigated (see Table 4, Design Key). All individualshad signed an informed consent form. The protocols for obtaining patientsamples were approved by the Institutional Review Board (IRB).

A second sample set was obtained from the Cleveland Clinic FoundationHeart Center (CCF), from individuals who had undergone diagnostic orinterventional cardiac catheterization. This set consisted of DNA fromCaucasian individuals who had had no coronary artery stenosis, orstenosis varying in degree from low to severe as evidenced by coronaryangiography. A subset of individuals also had a history of MI. Sampleswere divided into cases and controls: cases were individuals with ahistory of MI, while controls had no MI and varying levels of stenosis.For each particular SNP association study, a subset of the case andcontrol populations were investigated (see Table 4, Design Key). “Lowstenosis” was considered less than 50% stenosis by angiography.Individuals were between the ages of 18-75 and had signed an informedconsent form. The protocols for obtaining patient samples were approvedby the IRB.

DNA was extracted from blood samples at CCF or UCSF using conventionalDNA extraction methods or commercially available kits, such as theQIA-amp kit from Qiagen (Valencia, Calif.), according to themanufacturer's suggestions. SNP markers in the extracted DNA wereanalyzed by genotyping. Initially, pooling studies were performed inwhich DNA samples from about 50 individuals from each sample set werepooled and the allele frequencies for specific markers were obtainedusing a PRISM® 7900HT Sequence Detection System (Applied Biosystems,Foster City, Calif.) by allele-specific PCR, similar to the methoddescribed by Germer et al. Genome Research 10:258-266 (2000). (PCRprimers used in this example and Example 2, following, are shown inTable 3 with their corresponding SNP markers by hCV.) Those SNPs with anobserved P value<0.1 (Fisher Exact test) for association with MI in thepooled samples were selected as candidates for validation by individualgenotyping.

For validation of SNP association with MI based on the results of thepooling studies, samples from CCF and UCSF were individually genotypedby performing oligonucleotide ligation assays (OLA). Briefly, in OLA thegenomic regions containing the SNPs of interest were amplified fromsample DNA using upper and lower PCR primers. The resultant ampliconswere then mixed in a hybridization reaction with the two allele-specificoligonucleotides (ASO1 and ASO2) and one common ligation-specificoligonucleotide (LSO) for each marker. Each allele-specificoligonucleotide was attached to a Luminex® bead that had a specificfluorescence. One of the two allele-specific oligonucleotides hybridizedwith each amplicon, and was then ligated to the LSO by the ligase in thereaction mixture. The samples' genotypes were determined when theligated products were then detected in a Luminex®100™ fluorimeter(Luminex Corporation, Austin, Tex.).

Genotype or allele frequencies of SNPs in the UCSF and CCF samples wereanalyzed for association with MI as a clinical endpoint. Results of thisanalysis are reported in Table 4, for 44 SNPs showing association withCHD, specifically based on their association with MI. Allele or genotypefrequencies for the tested SNPs were compared between cases and controlsto determine MI risk association, according to the various case/controldesigns (Table 4). The magnitude of the allelic or genotypic effect onrisk association (effect size) was estimated by an odds ratio (OR). Anallele or genotype may be under- or overrepresented in cases. An alleleor genotype overrepresented in cases indicates that the reported alleleor genotype is a risk factor for disease. An allele or genotypeunderrepresented in cases indicates that this allele/genotype isprotective against disease. A SNP was considered a marker for MI risk ifthe association analyses in the two sample sets (CCF and UCSF) showedthe same risk allele, and P values less than or equal to 0.1 in bothsets. Allelic P values were calculated using the Fisher Exact test;genotypic (i.e. dominant/recessive) P values were calculated using theasymptotic chi-square test. No multiple testing corrections were made.Where the allele frequencies of cases vs. controls indicated an MI riskassociation in a particular population stratification, this is indicatedin the “Stratum” columns of Table 4. For example, hCV1283127 wasobserved to be associated with MI when allele frequencies in male MIcases were compared with male non-MI controls. Other populationstratifications where a particular SNP was found to be associated withMI were age, body mass index (BMI), hypertension, sex, and smokingstatus (smokers and non-smokers).

One example of a SNP marker where the genotype containing two copies ofthe risk allele is associated with an increased risk for MI (i.e.homzygous recessive) is hCV 16189747, a marker in the SNX19 gene (Table4). Samples from CCF and UCSF were individually genotyped forhCV16189747 (Table 4). In the CCF sample set, 446 cases and 577 controlswere genotyped. Cases all had a history of MI, while controls had no MIand low (<50%) or no stenosis by angiography. In the UCSF sample set,740 cases and 951 controls were genotyped. Cases had a history of MIwhile controls had no symptomatic CVD. Individuals with two copies ofthe C allele at this SNP position, i.e. inheritance mode recessive(Rec), in both CCF and UCSF sets showed a significant association withincreased risk of MI (P values of 0.093 and 0.056, OR of 1.24 and 1.21,respectively) when compared to those carrying one or no copies of therisk allele (heterozygous and homozygous for the non-risk allele), incase and control populations unstratified by phenotype (Stratum “All”).

This and all other SNP markers described herein may also be found inTables 1 and 2. In Table 1, context sequence information is providedregarding the transcript in which each SNP is found, if it resides in atranscript. For example this SNP marker, Celera SNP ID hCV 16189747, isfound in the transcript sequence of SEQ ID 217 (Table 1). Also providedin Table 1 is other information regarding the transcript in which theSNP is located: the gene symbol, SNX19; position of the SNP in thetranscript; chromosome, 11; the Public SNP ID (i.e. the rs, or RefSNP,number from the National Center for Biotechnology Information SNPdatabase if known), rs2298566; SNP type, Missense Mutation, etc. InTable 2 is provided genomic sequence information for all SNP markersdescribed herein. For example Celera SNP ID hCV16189747 is found in thegenomic sequence of SEQ ID 259 (Table 2). In the event that there areSNPs calculated to be in LD with the interrogated SNP, that informationis also provided in Tables 1 and 2, as a “Related Interrogated SNP.”E.g., see SEQ ID 393 in Table 2, where the Celera SNP ID for theinterrogated SNP is hCV3108698, and the related LD SNP directlyfollowing is hCV15954965 (power of 0.8).

Another example of a SNP marker where the genotype containing two copiesof the risk allele (homozygous) is associated with an increased risk forMI is hCV7425232, a marker in the MYH15 gene. Samples from CCF and UCSFwere individually genotyped for this SNP (Table 4). In the CCF sampleset, genotyping results were obtained from 170 female cases and 226female controls. Cases had a history of MI, while controls had no MI andlow (<50%) or no stenosis. In the UCSF sample set, genotyping resultsfrom 300 female cases and 553 female controls were obtained. Cases had ahistory of MI, while controls had no symptomatic CVD. An analysis of theallele frequencies obtained from the female cases vs. female controlsshowed an association of this SNP with MI in the female populationstratification (Stratum “FM”). Females with two copies of the C alleleat this SNP position, i.e. inheritance mode recessive (Rec), in both CCFand UCSF sets showed a significant association with increased risk of MI(P values of 0.049 and 0.029, OR of 1.90 and 1.69, respectively) whencompared to females carrying one or no copies of the risk allele(heterozygous and homozygous for the non-risk allele).

An example of a SNP marker where the genotype containing a single copyof the risk allele (i.e. heterozygous) is associated with an increasedrisk for MI is hCV9326822, a marker in the STX10 gene. Samples from CCFand UCSF were individually genotyped for this SNP (Table 4). In the CCFsample set, 398 cases and 233 controls were genotyped. Cases had ahistory of MI, while controls had no MI and no stenosis. In the UCSFsample set, 611 cases and 618 controls were genotyped. Cases had ahistory of MI, while controls had no symptomatic CVD. Individuals withone or two copies of the T allele of this marker, i.e. inheritance modedominant (Dom), in both CCF and UCSF sets showed a significantassociation with increased risk of MI (P values of 0.026 and 0.043, ORof 1.52 and 1.29, respectively) when compared to those carrying nocopies of the risk allele (homozygous for the non-risk allele), in caseand control populations unstratified by phenotype (Stratum “All”).

An example of a SNP where the risk allele is associated with anincreased risk of MI is hCV2091644, a marker in the VAMP8 gene. Samplesfrom CCF and UCSF were individually genotyped for hCV2091644 (Table 4).In the CCF sample set, 182 cases and 232 controls were genotyped. Caseshad a history of MI and were all <60 years old, while controls had noMI, low (<50%) or no stenosis, and were all >60 years old. In the UCSFsample set, 414 cases and 492 controls were genotyped. Cases had ahistory of MI and were either females<60 or males<55 years old, whilecontrols had no symptomatic CVD and were females>70 or males>65 yearsold. The C allele of this marker in both CCF and UCSF sets showed asignificant association with increased risk of MI (P values of 0.007 and0.017, OR of 1.48 and 1.26, respectively), in case and controlpopulations unstratified by phenotype (Stratum “All”).

A second case-control study for association with CHD, particularly MI,was conducted using the samples obtained from UCSF and CCF as describedabove. In this study three sequential genotyping analyses were performedin order to confirm the markers' association with MI.

In the first genotyping analysis, UCSF samples were divided into 340cases and 346 controls. DNA samples from about 50 individuals werepooled, and the pools genotyped for SNP markers by allele-specific PCR,described above. Those SNPs with an observed P value of <0.05 (FisherExact test) for association with MI in this analysis were selected ascandidates for follow-up genotyping in a second pooled-sample analysis.

In the subsequent genotyping analysis, DNA from the CCF sample set wasdivided into 445 cases and 606 controls. Cases were males under age 66and females under age 75 who had a history of MI verified byelectrocardiogram, cardiac enzymes or perfusion imaging. Controls had nohistory of MI and had less than 50% coronary luminal narrowing based onclinical angiography. Of the controls, 81% had stable angina. SNPs inthe pooled samples of this secondary analysis that demonstrated the samerisk allele as in the first, and had a P value of <0.05 for associationwith MI, were selected as candidates for individual genotyping in orderto further confirm their MI association.

UCSF DNA samples from 560 cases and 891 controls, males and females,were evaluated by individual genotyping. Subjects ranged in age from 52to 79. Controls had no history of MI, symptomatic vascular disease ordiabetes, and did not have a known first-degree relative with a historyof symptomatic coronary disease prior to age 65. Of the controls, 10%had undergone cardiac catheterization. Genotyping was performed usingOLA, as described above.

Each SNP was analyzed for association with MI by comparing SNP allele orgenotype frequencies between cases and controls. To determineassociation between MI and allele frequencies, the two-tailed FisherExact test was used for the first two pooled-sample analyses. Forindividually genotyped samples, where the a priori hypothesis was thatthe risk allele for the association matched the risk allele observed inthe previous two pooled-sample analyses, risk associations were assessedby the one-tailed Fisher Exact test. Genotypic comparisons ofhomozygotes to heterozygotes for the risk allele were performed usinglogistic regression, and the P values from logistic regression werecalculated using a Wald test. To correct for multiple testing, thevalues for false discovery rates, denoted here by Q_(i) for the i^(th)value in increasing order, were calculated using the MULTTEST procedure(SAS Institute, 2002; Benjamini and Hochberg, 1995) as follows: given mhypotheses H₁, H₂, . . . , H_(m) and corresponding P values P₁, P₂, . .. , P_(m), the P values are ordered such that P_((m))≧P_((m−1))≧ . . .≧P₍₁₎. Then let Q_((m))=P_((m)), Q_((m−1))=min(Q_((m)),P_((m−1))×[m÷(m−1)]), . . . , Q_((m−j))=min(Q_((m−j+1)),P_((m−j))×[m÷(m−j)]), . . . , Q₍₁₎=min(Q₍₂₎, m×P₍₁₎).

Five SNP markers are shown to be associated with MI in Tables 5 and 6.Table 5 lists each SNP marker's allelic association with MI, based onthe comparison of individual genotyping results from 560 cases and 891controls. Table 6 lists each SNP marker's genotypic association with MI.The number of cases and controls of each genotype that was analyzed foreach SNP marker is indicated. All listed markers demonstrated anassociation with increased risk of MI when the sample genotype washomozygous for the risk allele (Table 6, P values<0.05). A subset ofthese markers also demonstrated an association with increased MI riskwhen the genotype was heterozygous for the risk allele (Table 6, markersfor OR13G1 and ZNF627, P values<0.05).

An example of a SNP marker in which the risk allele provides anincreased risk of developing MI is hCV1449414, in the OR13G1 gene, witha P value of 0.013 (Table 5). The G allele is associated with anincreased risk of MI regardless of whether the individual's genotype washeterozygous or homozygous for the allele, with P values of 0.017 and0.019, respectively (Table 6).

A second example of a SNP marker in which the risk allele provides anincreased risk of developing MI is hCV25992024, in the ZNF627 gene, witha P value of 0.0034 (Table 5). The A allele is associated with anincreased risk of MI regardless of whether the individual's genotype washeterozygous or homozygous for this allele, with P values of 0.018 and0.002, respectively (Table 6).

An example of a SNP marker in which the association with MI risk isgreater for individuals with two copies of the risk allele thanindividuals with one (i.e. homozygotes vs. heterozygotes) ishCV12107274, in the TAS2R50 gene. Individuals with two copies of therisk allele C showed greater association with MI risk than individualswith one copy, with a P value of 0.007 for homozygotes (Table 6).

A second example of a SNP marker in which association with risk of MI isgreater for individuals with two copies of the risk allele than forindividuals with one is hCV11315168, in the ROS1 gene. Individuals withtwo copies of the risk allele C showed greater association with MI riskthan individuals with one, with a P value of 0.010 for homozygotes(Table 6).

Additional SNPs in five genes in which a SNP was shown to have anassociation with MI (Tables 4-6) were selected for further testing.These selected SNPs were individually genotyped in UCSF samples dividedinto cases and controls (see above for a description of this sampleset). Genotyping was performed by OLA or allele-specific PCR dependingon the assay, as described above. Based on the case and control allelefrequencies demonstrated in individual genotyping, the SNP markers inthese five genes that demonstrated an association with MI are shown inTable 7, with P values<0.1 and OR>1.0. Allelic P values were determinedusing the Fisher Exact test; genotypic (dominant/recessive) P values,using the asymptotic chi-square test.

The SNPs presented in Tables 4-7 are shown to be associated with riskfor CHD, specifically MI. Other SNPs (such as those presented in Tables1 and 2), including LD SNPs in the genes listed in Table 4 would also beexpected to be useful for the diagnosis, prognosis, etc., of CHD, andparticularly MI. All such SNPs associated with CHD may also be usefulfor predicting a patient's response to therapeutic agents such asstatins.

Example 2 Statistical Analysis of SNP Allelic and Genotypic Associationwith Stenosis Risk

A case-control genetic analysis was performed to determine theassociation of SNPs in the human genome with coronary heart disease(CHD), and in particular stenosis, using genomic DNA from twoindependently obtained sample sets. One set consisted of individualsfrom the Cleveland Clinic Foundation (CCF), divided into 782 cases and254 controls. Cases were individuals with the most severe stenosis, andcontrols were individuals with no detectable stenosis and no history ofMI. The second set of samples were obtained from the University ofCalifornia at San Francisco (UCSF). This set consisted of DNA from 770Caucasian individuals, divided into 472 cases and 298 controls. Caseswere individuals with the most severe stenosis, and controls wereindividuals with the least severe stenosis and no history of MI.Stenosis severity was determined in both sample sets by obtainingangiographic measurements of ten coronary artery segments perindividual, and ranking the individuals according to these measurements.DNA was extracted from blood samples at CCF or UCSF, as described abovein Example 1.

SNP markers in the extracted DNA samples were analyzed by genotyping.Initially, pooling studies were performed, in which DNA samples fromabout 50 individuals from each sample set were pooled and the allelefrequencies for specific markers were obtained by allele-specific PCR,as described in Example 1. SNP markers that were determined in poolingstudies to be significantly associated with stenosis were thenindividually genotyped by a similar method.

The CCF sample set was genotyped first, using pooled samples. SNPs withan observed P value<0.05 for association with stenosis and an oddsratio>1.3 were selected for further genotyping in the second set ofpooled samples, from UCSF. Those SNPs with stenosis association to thesame risk allele in both pooled-sample studies were individuallygenotyped in CCF and UCSF samples.

The SNP markers' association with stenosis was calculated, depending onwhether a genotype or allelic association was investigated: the FisherExact test for allelic association, and asymptotic chi-square test forgenotypic association of two different modes of inheritance (dominantand recessive). The magnitude of the allelic or genotypic effect on riskassociation (effect size) was estimated by the odds ratio (OR).

One validated SNP showing an association with CHD, specificallystenosis, is shown in Table 8. The SNP is considered a validated markerbecause the association analysis in individual genotyping indicated thesame risk allele as in the previous two pooled studies, with P values ofless than 0.05. The A risk allele for this SNP marker is associated withstenosis (P values of 0.04 in both sample sets). Additionally, the Aallele is dominant for stenosis risk; i.e., genotypic analysis indicatesassocation with stenosis when an individual is heterozygous for thisallele (Table 8, “Dom”).

The SNP marker presented in Table 8 was shown to be associated with riskfor CHD, specifically stenosis. Other SNP markers (such as thosepresented in Tables 1 and 2), including LD SNPs in the gene listed inTable 8 would also be expected to be useful for the diagnosis,prognosis, etc., of CHD, and particularly stenosis. All such SNPsassociated with CHD may also be useful for predicting a patient'sresponse to therapeutic agents such as statins.

Example 3 Additional SNPs in LD with CHD-Associated Interrogated SNPMarkers

An investigation was conducted to identify SNP markers in linkagedisequilibrium (LD) with SNPs which have been found to be associatedwith CHD, specifically MI and stenosis, as shown in Tables 4-8. Briefly,the power threshold (7) was set at 70% for detecting disease associationusing LD markers. This power threshold is based on equation (31) above,which incorporates allele frequency data from previous diseaseassociation studies, the predicted error rate for not detecting trulydisease-associated markers, and a significance level of 0.05. Using thispower calculation and the sample size, for each interrogated SNP athreshold level of LD, or r² value, was derived (r_(T) ², equations (32)and (33)). The threshold value r_(T) ² is the minimum value of linkagedisequilibrium between the interrogated SNP and its LD SNPs possiblesuch that the non-interrogated SNP still retains a power greater orequal to T for detecting disease-association.

Based on the above methodology, LD SNPs were found for all interrogatedSNPs shown in Tables 4-8. LD SNPs are listed in Table 9, each associatedwith its respective interrogated SNP. Also shown are the public SNP IDs(rs numbers) for interrogated and LD SNPs, the threshold r² value andthe power used to determine this, and the r² value of linkagedisequilibrium between the interrogated SNP and its matching LD SNP. Asan example in Table 9, MI-associated SNP hCV11315168 was calculated tobe in LD with hCV11315156 at an r² value of 0.71, based on a 70% powercalculation.

Two markers from Table 9 that were calculated to be in LD withCHD-associated SNPs were also analyzed for their association with MI(see the study description in Example 1, SNPs in five genes analyzed forMI association). SNP marker hCV11315171 is in calculated LD with markerhCV11315168, at an r² value of 1.00; hCV11315231 is in LD withhCV8908863, at an r² value of 0.63. Data for these two SNP markers areshown in Table 7 of Example 1. Both SNPs demonstrated an associationwith MI, with P values<0.05.

All publications and patents cited in this specification are hereinincorporated by reference in their entirety. Modifications andvariations of the described compositions, methods and systems of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments andcertain working examples, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the above-described modes for carryingout the invention that are obvious to those skilled in the field ofmolecular biology, genetics and related fields are intended to be withinthe scope of the following claims. TABLE 3 Sequence A Sequence BSequence C hCV Alleles (allele-specific primer) (allele-specific primer)(common primer) hCV1036123 C/T GTCCTACTGCCCCCG TGTCCTACTGCCCCCACCCCGGCTCCTCTTATAC (SEQ ID NO:505) (SEQ ID NO:506) (SEQ ID NO:507)hCV1116793 C/T GGAAGGTCATCCTGGG GGAAGGTCATCCTGGA CGAAGAGTTTCTGTGTGGTACAG(SEQ ID NO:508) (SEQ ID NO:509) (SEQ ID NO:510) hCV11247694 A/CCCTAGAGACTTAGGAAATGCTTT CCTAGAGACTTAGGAAATGCTTG GGTGGCAGGGTGAAACTCTA(SEQ ID NO:511) (SEQ ID NO:512) (SEQ ID NO:513) hCV11247713 A/GAGCTTAGGGAAAGGAGGAATAAA GCTTAGGGAAAGGAGGAATAAG GCGGCTGGGACCACTTT (SEQ IDNO:514) (SEQ ID NO:515) (SEQ ID NO:516) hCV11276368 C/TCGCGGAGTGTCAAGAGG CGCGGAGTGTCAAGAGA GCTGGCAGAGTGCTGTTGA (SEQ ID NO:517)(SEQ ID NO:518) (SEQ ID NO:519) hCV11315168 C/G GTTGTTTGCTTCATCTCTGCGTTGTTTGCTTCATCTCTGG AGTGCTGGGCTCAAGAAC (SEQ ID NO:520) (SEQ ID NO:521)(SEQ ID NO:522) hCV11315171 C/T ATTTCTGAATAACTGAAGTTGGTCATTTCTGAATAACTGAAGTTGGTT AAACTGGCAATAACTCAGATTCT (SEQ ID NO:523) (SEQ IDNO:524) (SEQ ID NO:525) hCV11315231 A/G CCCTTCTGCTTCACCTCTATCCCTTCTGCTTCACCTCTAC CTCCTACAACTCTTGCATTAGCATAAGA (SEQ ID NO:526) (SEQID NO:527) (SEQ ID NO:528) hCV11563866 C/G ATATTACAGTGGAAAAGAAGGGATATTACAGTGGAAAAGAAGGC ATTATGGCTGTTCTCAAACTTG (SEQ ID NO:529) (SEQ IDNO:530) (SEQ ID NO:531) hCV11987864 C/T CCAGCAGTGATGTGC ACCAGCAGTGATGTGTGCCTCAGCAGGACTTCTATG (SEQ ID NO:532) (SEQ ID NO:533) (SEQ ID NO:534)hCV12107274 C/T TGATGCTAATCTGTTCTCTGTG TCTGATGCTAATCTGTTCTCTGTAATAACAAGAGGAAGGAGATCAGA (SEQ ID NO:535) (SEQ ID NO:536) (SEQ ID NO:537)hCV1283127 C/G CAGGCATGACATTGAAAC CAGGCATGACATTGAAAGCAGAATGTAGTCTCGATTCTTCTTC (SEQ ID NO:538) (SEQ ID NO:539) (SEQ IDNO:540) hCV1328901 C/T GAGCAGTGAATCAGGTTAAC GAGCAGTGAATCAGGTTAATGGCCCTTGTTGGTGTCTTA (SEQ ID NO:541) (SEQ ID NO:542) (SEQ ID NO:543)hCV1449414 T/C ACACACATATGGTGGTTCATAAT CACACATATGGTGGTTCATAACCATGTCCCAGCTCTTCTTG (SEQ ID NO:544) (SEQ ID NO:545) (SEQ ID NO:546)hCV15752716 C/T ACGCTGCTGTTCCG ACGCTGCTGTTCCA CAGACAGACAACAATTCAGAAGAA(SEQ ID NO:547) (SEQ ID NO:548) (SEQ ID NO:549) hCV15770510 C/TACGGCATCTTCTATCCG CACGGCATCTTCTATCCA CCAGCTGGTGGTGAGTG (SEQ ID NO:550)(SEQ ID NO:551) (SEQ ID NO:552) hCV15807798 C/T GCTTGGAACTTTCCATGCGGCTTGGAACTTTCCATGT CCTTTAACAGTCCCTGCTACTT (SEQ ID NO:553) (SEQ IDNO:554) (SEQ ID NO:555) hCV15885108 A/C ATGAGCAGACTCTAAGCTGAGAGCAGACTCTAAGCTGC TGAGTGCCTGGGGAAGTT (SEQ ID NO:556) (SEQ ID NO:557)(SEQ ID NO:558) hCV15954965 A/G ATGCTCAAATGGGAAAGATGATTGCTCAAATGGGAAAGATGAC CATTACATTGCCTATCAGTGCAGTTAAGT (SEQ ID NO:559) (SEQID NO:560) (SEQ ID NO:561) hCV15965459 A/T CAGCCTCAGGAGATAACAGTCAGCCTCAGGAGATAACAGA TCAACCCTTTTCCACATAGAT (SEQ ID NO:562) (SEQ IDNO:563) (SEQ ID NO:564) hCV16189747 A/C ATGGACTCCTGAAGAAGCATGGACTCCTGAAGAAGCC TCAGTCCGCTGCCTTGTTAT (SEQ ID NO:565) (SEQ ID NO:566)(SEQ ID NO:567) hCV16196618 C/G ATTAGCCCCAAAGCGTAC ATTAGCCCCAAAGCGTAGGCTTTAGAAGGCTGGATATTTATG (SEQ ID NO:568) (SEQ ID NO:569) (SEQ ID NO:570)hCV1635402 A/G AACTCATCAAGATTTATGCTTCA ACTCATCAAGATTTATGCTTCGCTACTCAGGCCAGTCTTGTTACT (SEQ ID NO:571) (SEQ ID NO:572) (SEQ ID NO:573)hCV1639780 C/G GCCTACAGAAAGATTTCACAAAAC GCCTACAGAAAGATTTCACAAAAGAGGACTCTCAGACCCTTCCTAAATA (SEQ ID NO:574) (SEQ ID NO:575) (SEQ IDNO:576) hCV1690777 A/G GGCTTTACAGAAGGAAATGCT GCTTTACAGAAGGAAATGCCGCATGCGCTGAATTTTATATAG (SEQ ID NO:577) (SEQ ID NO:578) (SEQ ID NO:579)hCV1741111 C/T CCTCAGAATGGCCAAAAAC CCTCAGAATGGCCAAAAATCCAGGCAGCCAGACTTCT (SEQ ID NO:580) (SEQ ID NO:581) (SEQ ID NO:582)hCV1801149 A/G GAGAGTCGCAGGGTATTTTAA GAGAGTCGCAGGGTATTTTAGAAAGGCCCAGGCTCTAGA (SEQ ID NO:583) (SEQ ID NO:584) (SEQ ID NO:585)hCV1913911 C/G CTAGCAAAGCACACAAATGAC CTAGCAAAGCACACAAATGAGGGATGAAGTTTTTGAAACACAC (SEQ ID NO:586) (SEQ ID NO:587) (SEQ ID NO:588)hCV192122 C/T CTTTCTGTTTATCTGTGTTTCTTATC CTTTCTGTTTATCTGTGTTTCTTATTGCACAGCCATAATGAACAATA (SEQ ID NO:589) (SEQ ID NO:590) (SEQ ID NO:591)hCV202800 C/T GGCTATAATGGAATGAATCTTAACAG GGCTATAATGGAATGAATCTTAACAAGATGGATCAAGCACTTTACCAACTCA (SEQ ID NO:592) (SEQ ID NO:593) (SEQ IDNO:594) hCV2091601 C/G GGAATGGGTCAAGAATGTTCC GGAATGGGTCAAGAATGTTCGGGCCCAAGGCAAAGGATTTT (SEQ ID NO:595) (SEQ ID NO:596) (SEQ ID NO:597)hCV2091606 A/G CCCAGCCTATTCTTAATCCTATTTTA CCAGCCTATTCTTAATCCTATTTTGTCCTAGCAATTCCCTGTGTGATA (SEQ ID NO:598) (SEQ ID NO:599) (SEQ ID NO:600)hCV2091642 A/G CGTTCTTCCACCAGAATTTT CGTTCTTCCACCAGAATTTCGGGCTGCACTTGTACTCTTATAAT (SEQ ID NO:601) (SEQ ID NO:602) (SEQ ID NO:603)hCV2091643 C/T GGAGGGTTGTCCCTGAAG GGAGGGTTGTCCCTGAAAGGTGGAAGAACGTGAAGATGATTGT (SEQ ID NO:604) (SEQ ID NO:605) (SEQ IDNO:606) hCV2091644 C/T TTCTGGGGCATACAACG CTTCTGGGGCATACAACAAGGGACAACCCTCCATAAA (SEQ ID NO:607) (SEQ ID NO:608) (SEQ ID NO:609)hCV2091655 C/T AGAATGGCGTCTACCCAG AGAATGGCGTCTACCCAACAGGTAGCTTGGTTACCCATTCTT (SEQ ID NO:610) (SEQ ID NO:611) (SEQ ID NO:612)hCV2100197 C/T TGTGAAGATCCAAACCACTC TGTGAAGATCCAAACCACTTTGAGCCATCCATCTTCTACA (SEQ ID NO:613) (SEQ ID NO:614) (SEQ ID NO:615)hCV2100250 C/T GTATGTAGTAAATAGTTAATAAAGGA GGTATGTAGTAAATAGTTAATAAAGGAAAGATACTAGCCATATCATTACATGTGTG GTGATAC (SEQ ID NO:616) AGTGATAT (SEQ IDNO:617) (SEQ ID NO:618) hCV2169762 G/T CGAGTCGGTCTGCTGC CGAGTCGGTCTGCTGATGCCTACCTCATTCCATCTG (SEQ ID NO:619) (SEQ ID NO:620) (SEQ ID NO:621)hCV25605409 A/C CAAAACAGAAGAGCAAGATGA AAAACAGAAGAGCAAGATGCTTTTGGTTCGATGGATGTT (SEQ ID NO:622) (SEQ ID NO:623) (SEQ ID NO:624)hCV25609987 A/G GAGCAGGTAGCCTGTATTT GAGCAGGTAGCCTGTATTCTGCTGCCTTGGTTGTGA (SEQ ID NO:625) (SEQ ID NO:626) (SEQ ID NO:627)hCV25611853 G/T AGGACTGGATGAAATGAGTG AAGGACTGGATGAAATGAGTTGCCAGAGCAGAGTGACATCT (SEQ ID NO:628) (SEQ ID NO:629) (SEQ ID NO:630)hCV25623749 G/C CCAGAGAAGTCCAACGC CCAGAGAAGTCCAACGGGACCTCCCTGACCTACTTAGAC (SEQ ID NO:631) (SEQ ID NO:632) (SEQ ID NO:633)hCV25626077 C/T GGGTTTTGGTTCTTACCACAC GGGTTTTGGTTCTTACCACATGGCTGAGGTTCTCATCCAGTAA (SEQ ID NO:634) (SEQ ID NO:635) (SEQ ID NO:636)hCV25628370 C/T CTGAGACAAAATTGAGGAAGC GCTGAGACAAAATTGAGGAAGTTGAAACATCAAACAAGAAATCAGT (SEQ ID NO:637) (SEQ ID NO:638) (SEQ ID NO:639)hCV25641925 A/G GCGTGATCGCAAGAGTCT CGTGATCGCAAGAGTCCGGGTGTCTGCTGCATCTACT (SEQ ID NO:640) (SEQ ID NO:641) (SEQ ID NO:642)hCV25651109 C/G GGTCCTGCTTGATGCG AGGTCCTGCTTGATGCC CGACCATGGACATTCACAT(SEQ ID NO:643) (SEQ ID NO:644) (SEQ ID NO:645) hCV25928538 C/GTCCACTGTTTTTGAACGC TCCACTGTTTTTGAACGG TAATTGCAAGAATATTGAAAGACA (SEQ IDNO:646) (SEQ ID NO:647) (SEQ ID NO:648) hCV25935078 C/TCTTCCAGATCCTCTGTCTTG GCTTCCAGATCCTCTGTCTTA CCAGTGAAGGTGGAGGAAATGATC (SEQID NO:649) (SEQ ID NO:650) (SEQ ID NO:651) hCV25937808 A/GAGAAGCTGAAAGGACCACTT GAAGCTGAAAGGACCACTC CTTCTGGAATTTGGTTTCCTGTTTCTTAG(SEQ ID NO:652) (SEQ ID NO:653) (SEQ ID NO:654) hCV25951678 A/GAATGCAGCTGCTCAAAGA ATGCAGCTGCTCAAAGG GTTCCCGGGCTCACA (SEQ ID NO:655)(SEQ ID NO:656) (SEQ ID NO:657) hCV25959434 C/G CCAGGAGCCCTCCAAGCCAGGAGCCCTCCAAC GGCCTCTTCCACTCACTAAA (SEQ ID NO:658) (SEQ ID NO:659)(SEQ ID NO:660) hCV25965660 A/G GGCCTGGTGGAAGTGAT GGCCTGGTGGAAGTGACGGCTGACCGTCATCCAC (SEQ ID NO:661) (SEQ ID NO:662) (SEQ ID NO:663)hCV25992024 G/A TTACAGGGTCTTTCTCCACC TCTTACAGGGTCTTTCTCCACTAGAGAAACCCCATGAAAGTAAG (SEQ ID NO:664) (SEQ ID NO:665) (SEQ ID NO:666)hCV26490082 A/G GGCTTGTTCCTGTTGGAA GGCTTGTTCCTGTTGGAGTCAAGGTGGGAGAAAGAGATGT (SEQ ID NO:667) (SEQ ID NO:668) (SEQ ID NO:669)hCV26809148 A/G CATCATGGTGTTCTTGCCT ATCATGGTGTTCTTGCCCCATTATCTGAAATGTTTCATTGTAGA (SEQ ID NO:670) (SEQ ID NO:671) (SEQ IDNO:672) hCV2731349 A/G TGGCAAACCATGGGTA GGCAAACCATGGGTGTGGAACCCACTTTCTTTCTTAC (SEQ ID NO:673) (SEQ ID NO:674) (SEQ ID NO:675)hCV27484788 A/T GAAATGCTTGTTTCTTTCTAAATTG GAAATGCTTGTTTCTTTCTAAATTGCCAACAATAGAGAAGCAGACAAATATTC TA (SEQ ID NO:676) TT (SEQ ID NO:677) (SEQID NO:678) hCV28026155 C/G GAGGGAGACTTGGTTGTCC AGGGAGACTTGGTTGTCGCCTAATTAGGAAGCAAATTCATTC (SEQ ID NO:679) (SEQ ID NO:680) (SEQ ID NO:681)hCV29281059 G/T CTCATCTCTCTCTAGGCTAGTG CTCATCTCTCTCTAGGCTAGTTGGGACCGCTGGTCTATAGTAC (SEQ ID NO:682) (SEQ ID NO:683) (SEQ ID NO:684)hCV2930693 A/C CGAAGAAGTACAACCCACAT CGAAGAAGTACAACCCACAGGACACATGTAAGTTCCACTCATATG (SEQ ID NO:685) (SEQ ID NO:686) (SEQ IDNO:687) hCV30375335 A/G CACTCACTGGCATTTTCTTCTAT ACTCACTGGCATTTTCTTCTACCTTGTGGAAAGAAGTAGGGCAAATATCA (SEQ ID NO:688) (SEQ ID NO:689) (SEQ IDNO:690) hCV3049077 C/T CAAACCACTTAGTACCACTAGATGTCAAACCACTTAGTACCACTAGATA TTCTCATAAAGGCTCACCCAGGAAA (SEQ ID NO:691) (SEQID NO:692) (SEQ ID NO:693) hCV30555387 C/T GCTAAACTTATCTTTGACTGATTACGCTAAACTTATCTTTGACTGATTAC GGAGTTAGGAAGTGCTTTGCAGAAA AG (SEQ ID NO:694)AA (SEQ ID NO:695) (SEQ ID NO:696) hCV3108768 C/T TCCACTTGGTACTTGGGGATCCACTTGGTACTTGGGA CCGCTGTGGCAACTATGTACTAC (SEQ ID NO:697) (SEQ IDNO:698) (SEQ ID NO:699) hCV3108811 C/T GAAACTAAAAGGGTCAAGAATAGCGGAAACTAAAAGGGTCAAGAATAGT CCAACCCCAATACCACATAGTTT (SEQ ID NO:700) (SEQID NO:701) (SEQ ID NO:702) hCV31258057 G/T AGTGCAGGGTGGATGACAGTGCAGGGTGGATGAA CTCCTCTGGGGACTCTCAACT (SEQ ID NO:703) (SEQ ID NO:704)(SEQ ID NO:705) hCV3130332 A/G AGTTTATCTGTGCTCCCTAATTAAAGTTTATCTGTGCTCCCTAATTAG CCTGCTCCTTGCTGATG (SEQ ID NO:706) (SEQ IDNO:707) (SEQ ID NO:708) hCV3137630 A/C AACCTGAAAGCCACGTAGATACCTGAAAGCCACGTAGAG CTGTCCAGAAATCATTCATCAT (SEQ ID NO:709) (SEQ IDNO:710) (SEQ ID NO:711) hCV3180404 A/G ACAGCAGAGCAGCCTTAAACAGCAGAGCAGCCTTAG AGTGATGCTGGAAGCACTTCT (SEQ ID NO:712) (SEQ ID NO:713)(SEQ ID NO:714) hCV3181997 A/G TGGATCCTGACTTTGTGAAATTGGATCCTGACTTTGTGAAAC GGAATCTGAAGGAGACATTTTTAC (SEQ ID NO:715) (SEQ IDNO:716) (SEQ ID NO:717) hCV3225041 A/G AACAGGAGTTTGAACTTGGTTAACAGGAGTTTGAACTTGGTC CAGGGAAGGACGGCCTAATATCA (SEQ ID NO:718) (SEQ IDNO:719) (SEQ ID NO:720) hCV3225044 G/A ACGTGCTGGTGGGAG ACGTGCTGGTGGGAACCTCCTGGAGCAGCTATTC (SEQ ID NO:721) (SEQ ID NO:722) (SEQ ID NO:723)hCV323070 A/G GCCCAGAAAGATGAGTTCA GCCCAGAAAGATGAGTTCGTGTCCCTTTTTCAGAGACATAGAT (SEQ ID NO:724) (SEQ ID NO:725) (SEQ ID NO:726)hCV323071 A/G AAACCAGGATATCAGAACATTTTA ACCAGGATATCAGAACATTTTGGGTCTTAGGAATTATCTGACATCTT (SEQ ID NO:727) (SEQ ID NO:728) (SEQ IDNO:729) hCV49128 G/T CTTGGCCTTCTCAGGAAC CTTGGCCTTCTCAGGAAAAACATTCTGCCCATCACAGACATTC (SEQ ID NO:730) (SEQ ID NO:731) (SEQ IDNO:732) hCV505516 A/T CACCATGGTGAACACAAATAA CACCATGGTGAACACAAATATCCGCTCCAACCAACCTTTA (SEQ ID NO:733) (SEQ ID NO:734) (SEQ ID NO:735)hCV7422490 C/T GCATGAAACATGGGATTGTATAC AGCATGAAACATGGGATTGTATATACACTCCTGGCCCTTATTTATACTC (SEQ ID NO:736) (SEQ ID NO:737) (SEQ IDNO:738) hCV7425232 C/T TCAAAATTATTTCTTGCTACAGG GTCAAAATTATTTCTTGCTACAGATCCTCCAGCCTCTCATTC (SEQ ID NO:739) (SEQ ID NO:740) (SEQ ID NO:741)hCV7425242 C/T TGGATGGCTGTGATTACAAC TTGGATGGCTGTGATTACAATGGCATAGTTCAGCCAAGTTCT (SEQ ID NO:742) (SEQ ID NO:743) (SEQ ID NO:744)hCV81794 C/T TGTCATATATGGTTTATTTACTACA TGTCATATATGGTTTATTTACTACATGACCCAGCAACTGAGTGTA AC (SEQ ID NO:745) AT (SEQ ID NO:746) (SEQ IDNO:747) hCV8367080 C/T CCAGCCTTCCCTTTTTC CCAGCCTTCCCTTTTTTAGAGAGAAGGACACAATGTGAG (SEQ ID NO:748) (SEQ ID NO:749) (SEQ ID NO:750)hCV881283 C/G AGACACACAGGACACATG AGACACACAGGACACATC TTCTGCTCCCAGAACTAG(SEQ ID NO:751) (SEQ ID NO:752) (SEQ ID NO:753) hCV8908863 C/GCAAGCAAGTTTCTCAAAGATTATTTA AAGCAAGTTTCTCAAAGATTATTTATGGAAATTACTCCACCCTCTATTAGTG TTC (SEQ ID NO:754) TG (SEQ ID NO:755) (SEQID NO:756) hCV8908875 A/G CATTTGCTGTGCATGTGTTTA TTTGCTGTGCATGTGTTTGTGGGAAGATTTAACAAGGAATAAGAAGA (SEQ ID NO:757) (SEQ ID NO:758) (SEQ IDNO:759) hCV9302071 A/G TATTCATGAACACCAGGAATTAAAATTCATGAACACCAGGAATTAAAATC CTTACCACCCCTGGAAACACTTAGT TT (SEQ ID NO:760)(SEQ ID NO:761) (SEQ ID NO:762) hCV9326822 C/T CTCGGGACCAGTCCAGCTCGGGACCAGTCCAA CCGACAGCCGAGGAGA (SEQ ID NO:763) (SEQ ID NO:764) (SEQID NO:765) hCV945276 G/T CGCCACAAACACATACCTG CGCCACAAACACATACCTTCCGCTGCTTGGAACAG (SEQ ID NO:766) (SEQ ID NO:767) (SEQ ID NO:768)hDV69288445 A/C AAGCCCTGCACATTTTATCTT AGCCCTGCACATTTTATCTGGCAGTGAAAGCCTGCATTAGAGA (SEQ ID NO:769) (SEQ ID NO:770) (SEQ ID NO:771)hDV70327769 A/T TCAAGAGACCTATGTTGGAGT GTCAAGAGACCTATGTTGGAGACATTGTGGAAGGCTTTGGCTATCT (SEQ ID NO:772) (SEQ ID NO:773) (SEQ ID NO:774)hDV72090975 C/T CAAGGAGCACAGCATCTG ACAAGGAGCACAGCATCTACAAGCAGGAGAGAGTGTGTAACT (SEQ ID NO:775) (SEQ ID NO:776) (SEQ ID NO:777)hDV72090986 G/T GGGAGAGGGCAACATTC GGGGAGAGGGCAACATTACCTTAGACAACCGTTTCCAGAGTAGA (SEQ ID NO:778) (SEQ ID NO:779) (SEQ IDNO:780) hDV72091004 C/G AAACTAGCACGAGAAAAGGAG AAACTAGCACGAGAAAAGGACCAGCCAAGGCTGGTGACAC (SEQ ID NO:781) (SEQ ID NO:782) (SEQ ID NO:783)

TABLE 4 Risk CCF UCSF SNP Marker Al- De- P De- P Identifier Gene Symbollele sign Case Cont Stratum Mode val OR sign Case Cont Stratum Mode valOR hCV1116793 none C C 173 371 All Rec 0.07 1.42 B 489 414 All Rec 0.051.31 hCV11276368 MKI67 C G 460 590 All Rec 0.02 1.44 A 768 971 All Rec0.02 1.33 hCV11563866 OR2A25 G D 184 235 All Allelic 0.00 1.50 B 505 456All Allelic 0.06 1.19 hCV11987864 none T C 200 432 All Dom 0.02 1.16 B527 475 All Dom 0.00 1.18 hCV1283127 none C F 287 121 M Allelic 0.081.34 A 462 402 M Allelic 0.07 1.21 hCV1328901 SLC26A8 C F 459 243 AllAllelic 0.07 1.24 A 767 972 All Allelic 0.06 1.14 hCV15752716 HPS1 T F339 109 S+ Allelic 0.00 2.12 A 490 432 S+ Allelic 0.02 1.45 hCV15770510ALOX12B T G 459 588 All Allelic 0.04 1.27 A 687 936 All Allelic 0.061.19 hCV15807798 none C G 458 589 All Rec 0.10 1.57 A 762 936 AllAllelic 0.01 1.25 hCV15965459¹ ZNF350 A F 321 136 BM Allelic 0.09 1.47 A404 316 All Rec 0.05 6.36 hCV16189747 SNX19 C G 446 577 All Rec 0.091.24 A 740 951 All Rec 0.06 1.21 hCV16196618 EIF2AK2 C F 458 245 AllAllelic 0.06 1.81 A 764 973 All Allelic 0.00 1.74 hCV1635402 CTNNA3 A F449 240 All Rec 0.09 1.38 A 511 315 Y Rec 0.08 1.36 hCV1690777 NFIB A F290 132 BM Allelic 0.01 2.95 A 399 314 All Allelic 0.10 1.61 hCV1741111GJA4 C F 425 164 BP+ Rec 0.07 1.40 A 444 323 BP+ Allelic 0.07 1.23hCV1801149² MKI67 G G 415 569 All Rec 0.04 1.42 A 757 959 All Rec 0.011.36 hCV1913911 none G G 287 354 M Allelic 0.02 1.31 A 447 386 M Allelic0.00 1.34 hCV192122 none C G 278 352 M Dom 0.02 1.66 A 258 526 S− Dom0.07 1.44 hCV2091644 VAMP8 C D 182 232 All Allelic 0.01 1.48 B 414 492All Allelic 0.02 1.26 hCV2169762 HPSE2 T F 455 243 All Dom 0.00 1.61 A769 972 All Dom 0.02 1.25 hCV25605409 C10orf28 C G 286 358 M Dom 0.011.56 A 458 400 M Dom 0.03 1.36 hCV25609987 WDR31 G G 460 590 All Allelic0.07 1.42 A 764 939 All Allelic 0.02 1.37 hCV25611853 GBGT1 G F 475 268All Allelic 0.09 1.49 A 793 1000 All Allelic 0.08 1.30 hCV25623749 PIGXG G 415 569 All Allelic 0.00 1.64 A 757 923 All Allelic 0.04 1.24hCV25628370 none C F 475 268 All Rec 0.10 1.20 A 793 1000 All Rec 0.011.22 hCV25641925 KCNK13 A E 287 605 M Dom 0.04 1.61 A 461 402 M Dom 0.051.52 hCV25651109 SLC39A7 G G 460 590 All Rec 0.04 1.62 A 764 933 All Rec0.06 1.37 hCV25928538 none G G 459 590 All Allelic 0.02 1.51 A 768 973All Allelic 0.00 1.60 hCV25951678 FCRLM2 A F 460 246 All Allelic 0.011.80 A 767 973 All Allelic 0.03 1.32 hCV25959434 FLJ42486 C G 475 619All Dom 0.08 >1 A 793 1000 All Dom 0.08 3.53 hCV25965660 MYOM3 A F 458244 All Dom 0.01 5.14 A 768 971 All Allelic 0.07 1.20 hCV2731349 none AG 287 357 M Dom 0.09 1.42 A 313 645 BP− Dom 0.10 1.32 hCV2930693 none AC 185 404 All Allelic 0.04 1.37 B 202 270 FM Allelic 0.04 1.40hCV3137630 VTI1A A E 285 605 M Dom 0.01 1.51 A 756 911 All Dom 0.03 1.28hCV3180404 COG2 A G 475 619 All Dom 0.08 1.28 A 793 1000 All Dom 0.081.14 hCV3181997 none A G 414 567 All Allelic 0.04 1.22 A 757 964 AllAllelic 0.06 1.14 hCV3185278³ XRRA1 C F 458 246 All Allelic 0.08 1.25 A765 939 All Allelic 0.02 1.21 hCV7425232 MYH15 C G 170 226 FM Rec 0.051.90 A 300 553 FM Rec 0.03 1.69 hCV81794 TMPRSS11B T F 435 235 All Rec0.01 3.52 A 658 802 All Allelic 0.02 1.23 hCV8367080 SGIP1 T F 171 123FM Rec 0.02 4.13 A 785 980 All Rec 0.00 1.64 hCV881283 PRKG1 G G 459 589All Allelic 0.03 1.72 A 769 975 All Allelic 0.04 1.42 hCV9326822 STX10 TF 398 233 All Dom 0.03 1.52 A 611 618 All Dom 0.04 1.29 hCV945276 KRT5 TD 183 232 All Rec 0.08 1.52 B 294 183 All Rec 0.04 1.67 hCV9626088⁴ noneA G 455 588 All Allelic 0.03 1.24 A 767 959 All Allelic 0.02 1.19¹hCV15965459 and hCV22274416 are directed to identical SNP markers.²hCV1801149 and hCV25473208 are directed to identical SNP markers.³hCV3185278 and hCV28026155 are directed to identical SNP markers.⁴hCV9626088 and hCV26809148 are directed to identical SNP markers.Stratum Key: BM BMI >= 27 kg/m² BP+ Hypertension BP− No hypertension FMFemale M Male S+ Smoking S− Nonsmoking Y Age <60 Design Key: A Caseswith MI vs. controls with no CVD B Younger cases with MI (female <60,male <55) vs. Older controls with no CVD (female >70, male >65) CYounger cases with MI (<60) vs. Older controls with no MI (>60) DYounger cases with MI (<60) vs. Older cases with no MI and low or nostenosis (>60) E Cases with MI vs. Controls with no MI F Cases with MIvs. Controls with no MI and no stenosis G Cases with MI vs. Controlswith no MI and low or no stenosis

TABLE 5 SNP Marker Risk OR Gene Symbol Identifier Allele P value OR 90%CI KIAA0992 hCV323070 G 0.0028 1.25 1.10-1.43 (palladin) ROS1hCV11315168 C 0.0120 1.23 1.06-1.42 TAS2R50 hCV12107274 C 0.0018 1.281.11-1.46 OR13G1 hCV1449414 G 0.0130 1.19 1.05-1.36 ZNF627 hCV25992024 A0.0034 1.25 1.09-1.44

TABLE 6 SNP Marker Gene Symbol Identifier Genotype Cases Controls OR Pvalue KIAA0992 (palladin) hCV323070 AA 73 132 GA 233 430 0.98 0.470 GG253 326 1.40 0.024 ROS1 hCV11315168 GG 298 522 GC 213 324 1.15 0.110 CC40 40 1.75 0.010 TAS2R50 hCV12107274 TT 54 116 CT 226 396 1.23 0.140 CC273 372 1.58 0.007 OR13G1 hCV1449414 AA 154 293 AG 286 416 1.31 0.017 GG113 154 1.40 0.019 ZNF627 hCV25992024 GG 49 118 AG 224 359 1.50 0.018 AA285 405 1.69 0.002

TABLE 7 SNP Marker Gene Risk Identifier Symbol Allele Design CasesControls Mode P value OR hCV15885108 MYH15 A B 500 459 Dom 0.051 1.4hCV1639780 MYH15 G A 762 972 Dom 0.082 1.2 hCV202800 MYH15 T A 767 977Allelic 0.006 1.2 hCV25937808 MYH15 G A 768 974 Rec 0.059 3.2hCV29281059 MYH15 G B 497 458 Dom 0.039 1.7 hCV30375335 MYH15 G B 501456 Dom 0.006 1.6 hCV3049077 MYH15 C B 500 460 Dom 0.033 1.7 hCV30555387MYH15 C B 504 461 Rec 0.084 1.8 hCV7422490 MYH15 T A 768 973 Allelic0.033 1.5 hCV7425242 MYH15 C B 501 460 Rec 0.098 1.3 hCV9302071 MYH15 AB 502 458 Rec 0.024 1.3 hDV69288445 MYH15 C A 765 977 Rec 0.046 1.3hDV70327769 MYH15 T A 767 973 Dom 0.088 1.6 hCV11247694 PALLD C A 762973 Allelic 0.006 1.2 hCV11247713 PALLD A A 759 967 Rec 0.055 1.2hCV323071 PALLD A A 751 963 Allelic 0.004 1.2 hCV49128 PALLD T A 651 819Rec 0.028 1.3 hCV505516 PALLD A A 758 972 Allelic 0.056 1.2 hDV72090975PALLD C B 502 460 Rec 0.094 1.6 hDV72090986 PALLD T A 763 974 Rec 0.0041.5 hDV72091004 PALLD G A 767 975 Allelic 0.020 1.6 hCV11315171 ROS1 T A690 835 Allelic 0.002 1.3 hCV11315231 ROS1 G A 763 971 Allelic 0.020 1.2hCV2100197 ROS1 C A 769 976 Dom 0.055 4.0 hCV2100250 ROS1 C A 763 975Dom 0.048 1.9 hCV27484788 ROS1 A A 766 974 Allelic 0.014 1.3 hCV8908863ROS1 C A 764 971 Allelic 0.000 1.3 hCV8908875 ROS1 A A 766 970 Allelic0.010 1.2 hCV15954965 SNX19 A A 762 976 Rec 0.003 1.4 hCV25626077 SNX19C A 767 976 Rec 0.009 1.3 hCV26490082 SNX19 A A 765 974 Rec 0.004 1.5hCV3108768 SNX19 T B 501 461 Allelic 0.061 1.2 hCV3108811 SNX19 T A 741970 Dom 0.046 1.2 hCV31258057 SNX19 T A 766 975 Allelic 0.070 1.1hCV1036123 VAMP8 C B 431 498 Rec 0.002 2.0 hCV2091601 VAMP8 G B 430 498Rec 0.057 4.1 hCV2091606 VAMP8 G B 430 498 Rec 0.057 4.1 hCV2091642VAMP8 A B 430 495 Allelic 0.016 1.3 hCV2091643 VAMP8 C B 429 498 Allelic0.019 1.3 hCV2091655 VAMP8 C B 428 485 Dom 0.065 1.3 hCV25935078 VAMP8 CB 430 498 Rec 0.010 1.9 hCV3225041 VAMP8 G B 431 498 Rec 0.011 1.8hCV3225044 VAMP8 G B 426 494 Rec 0.014 1.8 Design Key: A Cases with MIvs. controls with no CVD B Younger cases with MI (female <60, male <55)vs. Older controls with no CVD (female >70, male >65)

TABLE 8 SNP Marker Gene Risk Sample P OR 1 Sample P OR 2 IdentifierSymbol Allele Set 1 Mode 2 value 1 OR 1 95% Cl Set 2 Mode 2 value 2 OR 295% CI hCV3130332 K6IRS4 A CCF Allelic 0.04 1.32 0.98-1.70 UCSF Allelic0.04 1.28 0.99-1.63 CCF Dom 0.03 1.32 0.98-1.70 UCSF Dom 0.03 1.280.99-1.63 CCF Rec 0.30 1.32 0.98-1.70 UCSF Rec 0.42 1.28 0.99-1.63

TABLE 9 Interrogated Interrogated Threshold SNP rs LD SNP LD SNP rsPower r² r² hCV11315168 rs619203 hCV11315156 rs13197910 0.7 0.605 0.71hCV11315168 rs619203 hCV11315160 rs511764 0.7 0.605 1.00 hCV11315168rs619203 hCV11315160 rs511764 0.8 0.769 1.00 hCV11315168 rs619203hCV11315167 rs554017 0.7 0.605 0.71 hCV11315168 rs619203 hCV11315170rs529156 0.7 0.605 1.00 hCV11315168 rs619203 hCV11315170 rs529156 0.80.769 1.00 hCV11315168 rs619203 hCV11315171 rs529038 0.7 0.605 1.00hCV11315168 rs619203 hCV11315171 rs529038 0.8 0.769 1.00 hCV11315168rs619203 hCV11315172 rs526306 0.7 0.605 0.71 hCV11315168 rs619203hCV2217381 rs507280 0.7 0.605 0.63 hCV11315168 rs619203 hCV2217382rs587575 0.7 0.605 0.63 hCV11315168 rs619203 hCV2217394 rs483223 0.70.605 1.00 hCV11315168 rs619203 hCV2217394 rs483223 0.8 0.769 1.00hCV11315168 rs619203 hCV850544 rs485768 0.7 0.605 1.00 hCV11315168rs619203 hCV850544 rs485768 0.8 0.769 1.00 hCV11315171 rs529038hCV11315156 rs13197910 0.7 0.626 0.71 hCV11315171 rs529038 hCV11315160rs511764 0.7 0.626 1.00 hCV11315171 rs529038 hCV11315160 rs511764 0.80.796 1.00 hCV11315171 rs529038 hCV11315167 rs554017 0.7 0.626 0.71hCV11315171 rs529038 hCV11315168 rs619203 0.7 0.626 1.00 hCV11315171rs529038 hCV11315168 rs619203 0.8 0.796 1.00 hCV11315171 rs529038hCV11315170 rs529156 0.7 0.626 1.00 hCV11315171 rs529038 hCV11315170rs529156 0.8 0.796 1.00 hCV11315171 rs529038 hCV11315172 rs526306 0.70.626 0.71 hCV11315171 rs529038 hCV2217381 rs507280 0.7 0.626 0.63hCV11315171 rs529038 hCV2217382 rs587575 0.7 0.626 0.63 hCV11315171rs529038 hCV2217394 rs483223 0.7 0.626 1.00 hCV11315171 rs529038hCV2217394 rs483223 0.8 0.796 1.00 hCV11315171 rs529038 hCV850544rs485768 0.7 0.626 1.00 hCV11315171 rs529038 hCV850544 rs485768 0.80.796 1.00 hCV15807798 rs2486758 hCV29348349 rs6584535 0.7 0.899 1.00hCV15807798 rs2486758 hCV30611452 rs9420881 0.7 0.899 1.00 hCV15954965rs2236711 hCV12036362 rs1893019 0.7 0.780 0.97 hCV15954965 rs2236711hCV12036363 rs1893018 0.7 0.780 1.00 hCV15954965 rs2236711 hCV12036363rs1893018 0.8 0.992 1.00 hCV15954965 rs2236711 hCV12036366 rs1893017 0.70.780 1.00 hCV15954965 rs2236711 hCV12036366 rs1893017 0.8 0.992 1.00hCV15954965 rs2236711 hCV15956925 rs2282579 0.7 0.780 0.93 hCV15954965rs2236711 hCV16140214 rs2155752 0.7 0.780 1.00 hCV15954965 rs2236711hCV16140214 rs2155752 0.8 0.992 1.00 hCV15954965 rs2236711 hCV16140215rs2155751 0.7 0.780 1.00 hCV15954965 rs2236711 hCV16140215 rs2155751 0.80.992 1.00 hCV15954965 rs2236711 hCV25626063 rs3751039 0.7 0.780 0.93hCV15954965 rs2236711 hCV26490015 rs7942621 0.7 0.780 1.00 hCV15954965rs2236711 hCV26490015 rs7942621 0.8 0.992 1.00 hCV15954965 rs2236711hCV27467549 rs3190331 0.7 0.780 1.00 hCV15954965 rs2236711 hCV27467549rs3190331 0.8 0.992 1.00 hCV15954965 rs2236711 hCV27497392 rs3829271 0.70.780 0.84 hCV15954965 rs2236711 hCV27868998 rs4936121 0.7 0.780 0.93hCV15954965 rs2236711 hCV27869000 rs4436551 0.7 0.780 1.00 hCV15954965rs2236711 hCV27869000 rs4436551 0.8 0.992 1.00 hCV15954965 rs2236711hCV27869001 rs4264159 0.7 0.780 1.00 hCV15954965 rs2236711 hCV27869001rs4264159 0.8 0.992 1.00 hCV15954965 rs2236711 hCV27931232 rs4456262 0.70.780 1.00 hCV15954965 rs2236711 hCV27931232 rs4456262 0.8 0.992 1.00hCV15954965 rs2236711 hCV27996234 rs4457753 0.7 0.780 1.00 hCV15954965rs2236711 hCV27996234 rs4457753 0.8 0.992 1.00 hCV15954965 rs2236711hCV29138820 rs7119425 0.7 0.780 0.83 hCV15954965 rs2236711 hCV29138826rs4936123 0.7 0.780 1.00 hCV15954965 rs2236711 hCV29138826 rs4936123 0.80.992 1.00 hCV15954965 rs2236711 hCV29138827 rs6590520 0.7 0.780 1.00hCV15954965 rs2236711 hCV29138827 rs6590520 0.8 0.992 1.00 hCV15954965rs2236711 hCV3108698 rs1054869 0.7 0.780 1.00 hCV15954965 rs2236711hCV3108698 rs1054869 0.8 0.992 1.00 hCV15954965 rs2236711 hCV3108699rs10160281 0.7 0.780 1.00 hCV15954965 rs2236711 hCV3108699 rs101602810.8 0.992 1.00 hCV15954965 rs2236711 hCV31258081 rs10791100 0.7 0.7801.00 hCV15954965 rs2236711 hCV31258081 rs10791100 0.8 0.992 1.00hCV15954965 rs2236711 hCV31258087 rs10894273 0.7 0.780 1.00 hCV15954965rs2236711 hCV31258087 rs10894273 0.8 0.992 1.00 hCV15954965 rs2236711hCV31258089 rs12363140 0.7 0.780 1.00 hCV15954965 rs2236711 hCV31258089rs12363140 0.8 0.992 1.00 hCV15954965 rs2236711 hCV31258092 rs112223690.7 0.780 1.00 hCV15954965 rs2236711 hCV31258092 rs11222369 0.8 0.9921.00 hCV15954965 rs2236711 hCV31258095 rs12365680 0.7 0.780 0.84hCV15954965 rs2236711 hCV31258101 rs4459316 0.7 0.780 1.00 hCV15954965rs2236711 hCV31258101 rs4459316 0.8 0.992 1.00 hCV15954965 rs2236711hCV31258104 rs10791103 0.7 0.780 1.00 hCV15954965 rs2236711 hCV31258104rs10791103 0.8 0.992 1.00 hCV15954965 rs2236711 hCV31258105 rs71069730.7 0.780 0.96 hCV15954965 rs2236711 hCV31258108 rs7107595 0.7 0.7801.00 hCV15954965 rs2236711 hCV31258108 rs7107595 0.8 0.992 1.00hCV15954965 rs2236711 hCV7507666 rs1050078 0.7 0.780 0.84 hCV15954965rs2236711 hDV71141362 rs876641 0.7 0.780 1.00 hCV15954965 rs2236711hDV71141362 rs876641 0.8 0.992 1.00 hCV16196618 rs2307469 hCV25927064rs10202693 0.7 0.780 1.00 hCV16196618 rs2307469 hCV25927064 rs102026930.8 0.992 1.00 hCV16196618 rs2307469 hCV2787341 rs10176233 0.7 0.7801.00 hCV16196618 rs2307469 hCV2787341 rs10176233 0.8 0.992 1.00hCV16196618 rs2307469 hCV2787344 rs12469391 0.7 0.780 1.00 hCV16196618rs2307469 hCV2787344 rs12469391 0.8 0.992 1.00 hCV16196618 rs2307469hCV2787347 rs12470942 0.7 0.780 1.00 hCV16196618 rs2307469 hCV2787347rs12470942 0.8 0.992 1.00 hCV16196618 rs2307469 hCV30087629 rs46481970.7 0.780 1.00 hCV16196618 rs2307469 hCV30087629 rs4648197 0.8 0.9921.00 hCV16196618 rs2307469 hCV30177777 rs9631047 0.7 0.780 1.00hCV16196618 rs2307469 hCV30177777 rs9631047 0.8 0.992 1.00 hCV16196618rs2307469 hCV31844737 rs4648203 0.7 0.780 1.00 hCV16196618 rs2307469hCV31844737 rs4648203 0.8 0.992 1.00 hCV16196618 rs2307469 hCV31844752rs4648228 0.7 0.780 1.00 hCV16196618 rs2307469 hCV31844752 rs4648228 0.80.992 1.00 hCV16196618 rs2307469 hCV31844783 rs13424255 0.7 0.780 1.00hCV16196618 rs2307469 hCV31844783 rs13424255 0.8 0.992 1.00 hCV16196618rs2307469 hCV31844785 rs13398178 0.7 0.780 1.00 hCV16196618 rs2307469hCV31844785 rs13398178 0.8 0.992 1.00 hCV16196618 rs2307469 hCV31844790rs13426400 0.7 0.780 1.00 hCV16196618 rs2307469 hCV31844790 rs134264000.8 0.992 1.00 hCV16196618 rs2307469 hCV32378670 rs4648182 0.7 0.7801.00 hCV16196618 rs2307469 hCV32378670 rs4648182 0.8 0.992 1.00hCV1913911 rs2477037 hCV11646539 rs2249547 0.7 0.749 0.96 hCV1913911rs2477037 hCV11646539 rs2249547 0.8 0.953 0.96 hCV1913911 rs2477037hCV11646540 rs2249473 0.7 0.749 0.96 hCV1913911 rs2477037 hCV11646540rs2249473 0.8 0.953 0.96 hCV1913911 rs2477037 hCV15931271 rs2249834 0.70.749 0.90 hCV1913911 rs2477037 hCV1913874 rs960875 0.7 0.749 0.84hCV1913911 rs2477037 hCV1913878 rs2254342 0.7 0.749 0.87 hCV1913911rs2477037 hCV1913893 rs2243537 0.7 0.749 0.87 hCV1913911 rs2477037hCV1913894 rs912491 0.7 0.749 0.86 hCV1913911 rs2477037 hCV1913897rs912488 0.7 0.749 0.97 hCV1913911 rs2477037 hCV1913897 rs912488 0.80.953 0.97 hCV1913911 rs2477037 hCV1913899 rs2494983 0.7 0.749 0.90hCV1913911 rs2477037 hCV1913900 rs2246164 0.7 0.749 0.96 hCV1913911rs2477037 hCV1913900 rs2246164 0.8 0.953 0.96 hCV1913911 rs2477037hCV3120077 rs722569 0.7 0.749 0.90 hCV1913911 rs2477037 hCV3120083rs2249457 0.7 0.749 0.96 hCV1913911 rs2477037 hCV3120083 rs2249457 0.80.953 0.96 hCV1913911 rs2477037 hCV3120088 rs2255751 0.7 0.749 0.90hCV1913911 rs2477037 hCV3120090 rs2477035 0.7 0.749 0.97 hCV1913911rs2477037 hCV3120090 rs2477035 0.8 0.953 0.97 hCV1913911 rs2477037hCV8881174 rs1359282 0.7 0.749 0.90 hCV1913911 rs2477037 hCV8881179rs1007499 0.7 0.749 0.90 hCV1913911 rs2477037 hCV8881199 rs1324314 0.70.749 0.90 hCV1913911 rs2477037 hDV71123422 rs2477036 0.7 0.749 0.97hCV1913911 rs2477037 hDV71123422 rs2477036 0.8 0.953 0.97 hCV202800rs3996003 hCV27977948 rs4273372 0.7 0.801 0.84 hCV202800 rs3996003hDV71161241 rs7433826 0.7 0.801 0.81 hCV8908863 rs1535330 hCV11315231rs3798381 0.7 0.461 0.63 hCV8908863 rs1535330 hCV11315231 rs3798381 0.80.586 0.63 hCV8908863 rs1535330 hCV15989251 rs2354341 0.7 0.461 0.52hCV8908863 rs1535330 hCV16176657 rs2243383 0.7 0.461 0.60 hCV8908863rs1535330 hCV16176657 rs2243383 0.8 0.586 0.60 hCV8908863 rs1535330hCV2100216 rs3798385 0.7 0.461 0.58 hCV8908863 rs1535330 hCV2100217rs3798384 0.7 0.461 0.66 hCV8908863 rs1535330 hCV2100217 rs3798384 0.80.586 0.66 hCV8908863 rs1535330 hCV2100221 rs9481704 0.7 0.461 0.58hCV8908863 rs1535330 hCV2100223 rs9374655 0.7 0.461 1.00 hCV8908863rs1535330 hCV2100223 rs9374655 0.8 0.586 1.00 hCV8908863 rs1535330hCV2100227 rs3798383 0.7 0.461 0.63 hCV8908863 rs1535330 hCV2100227rs3798383 0.8 0.586 0.63 hCV8908863 rs1535330 hCV2100230 rs9320597 0.70.461 0.63 hCV8908863 rs1535330 hCV2100230 rs9320597 0.8 0.586 0.63hCV8908863 rs1535330 hCV27424306 rs9401001 0.7 0.461 0.63 hCV8908863rs1535330 hCV27424306 rs9401001 0.8 0.586 0.63 hCV8908863 rs1535330hCV30502071 rs9401000 0.7 0.461 0.63 hCV8908863 rs1535330 hCV30502071rs9401000 0.8 0.586 0.63 hCV8908863 rs1535330 hCV3228481 rs2243384 0.70.461 0.56 hCV8908863 rs1535330 hDV71169900 rs2038497 0.7 0.461 0.62hCV8908863 rs1535330 hDV71169900 rs2038497 0.8 0.586 0.62 hCV8908875rs1321807 hCV16176657 rs2243383 0.7 0.916 1.00 hCV8908875 rs1321807hCV2100227 rs3798383 0.7 0.916 0.97 hCV8908875 rs1321807 hCV2100230rs9320597 0.7 0.916 0.97 hCV8908875 rs1321807 hCV27424306 rs9401001 0.70.916 0.97 hCV8908875 rs1321807 hCV30502071 rs9401000 0.7 0.916 0.97hCV8908875 rs1321807 hDV71169900 rs2038497 0.7 0.916 0.97

1. A method for identifying an individual who has an altered risk fordeveloping CHD, comprising detecting a single nucleotide polymorphism(SNP) in any one of the nucleotide sequences of SEQ ID NOS: 1-67 and135-504 in said individual's nucleic acids, wherein the SNP is asspecified in Table 1 and Table 2, respectively, and the presence of theSNP is correlated with an altered risk for MI in said individual.
 2. Themethod of claim 1 in which the altered risk is an increased risk.
 3. Themethod of claim 1 in which the altered risk is a decreased risk.
 4. Themethod of claim 1 in which detection is carried out by a processselected from the group consisting of: allele-specific probehybridization, allele-specific primer extension, allele-specificamplification, sequencing, 5′ nuclease digestion, molecular beaconassay, oligonucleotide ligation assay, size analysis, andsingle-stranded conformation polymorphism.
 5. An isolated nucleic acidmolecule comprising at least 8 contiguous nucleotides wherein one of thenucleotides is a single nucleotide polymorphism (SNP) selected from anyone of the nucleotide sequences in SEQ ID NOS: 1-67 and 135-504, or acomplement thereof, wherein the SNP is as specified in Table 1 and Table2, respectively.
 6. An isolated nucleic acid molecule that encodes anyone of the amino acid sequences in SEQ ID NOS: 68-134.
 7. An isolatedpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 68-134.
 8. An antibody that specifically bindsto a polypeptide of claim 7, or an antigen-binding fragment thereof. 9.An amplified polynucleotide containing a single nucleotide polymorphism(SNP) selected from any one of the nucleotide sequences of SEQ ID NOS:1-67 and 135-504, or a complement thereof, wherein the amplifiedpolynucleotide is between about 16 and about 1,000 nucleotides inlength.
 10. The amplified polynucleotide of claim 9 in which thenucleotide sequence comprises any one of the nucleotide sequences of SEQID NOS: 1-67 and 135-504, wherein the SNP is as specified in Table 1 andTable 2, respectively.
 11. An isolated polynucleotide which specificallyhybridizes to a nucleic acid molecule containing a single nucleotidepolymorphism (SNP) in any one of the nucleotide sequences in SEQ ID NOS:1-67 and 135-504, wherein the SNP is as specified in Table 1 and Table2, respectively.
 12. The polynucleotide of claim 11 that is 8-70nucleotides in length.
 13. The polynucleotide of claim 11 that is anallele-specific probe.
 14. The polynucleotide of claim 11 that is anallele-specific primer.
 15. The polynucleotide of claim 11, wherein thepolynucleotide comprises a nucleotide sequence selected from the groupconsisting of the primer sequences set forth in Table 3 (SEQ ID NOS:505-783).
 16. A kit for detecting a single nucleotide polymorphism (SNP)in a nucleic acid, comprising the polynucleotide of claim 11, a buffer,and an enzyme.
 17. A method of detecting a variant polypeptide,comprising contacting a reagent with a variant polypeptide encoded by anucleotide sequence containing a single nucleotide polymorphism (SNP) inany one of the nucleotide sequences of SEQ ID NOS: 1-67 and 135-504 in atest sample, wherein the SNP is as specified in Table 1 and Table 2,respectively, and detecting the binding of the reagent to thepolypeptide.
 18. A method for identifying an agent useful intherapeutically or prophylactically treating MI, comprising contactingthe polypeptide of claim 7 with a candidate agent under conditionssuitable to allow formation of a binding complex between the polypeptideand the candidate agent, and detecting the formation of the bindingcomplex, wherein the presence of the complex identifies said agent. 19.A method for identifying an individual who is in need of receivingtreatment for MI, comprising detecting a single nucleotide polymorphism(SNP) in a sample from said individual in any one of the nucleic acidsequences of SEQ ID NOS: 1-67 and 135-504, wherein the SNP is asspecified in Table 1 and Table 2, respectively, and treating thatindividual with a therapeutic agent.
 20. The method of claim 19, whereinthe therapeutic agent is a statin.